Melanocortin receptor-specific peptides

ABSTRACT

The invention relates to melanocortin receptor-specific cyclic peptides of Formula (I) 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, where R 1 , R 2 , R 3 , R 4a , R 4b , R 4c , R 5 , x and y are as defined in the specification. These compounds are particularly useful in the treatments of energy homeostasis and metabolism related (e.g. diabetes), food intake related and/or energy balance and body weight related diseases, disorders and/or conditions, including obesity, overweight and diseases, disorders and/or conditions associated with obesity and/or overweight, such as type 2 diabetes and metabolic syndrome.

TECHNICAL FIELD

The present invention relates to melanocortin receptor-specific cyclicpeptides which may be used in the treatment of melanocortinreceptor-mediated diseases, indications, conditions and syndromes, inparticular energy homeostasis and metabolism related (e.g. diabetes),food intake related and/or energy balance and body weight relateddiseases, disorders and/or conditions, including obesity, overweight anddiseases, disorders and/or conditions associated with obesity and/oroverweight, such as type 2 diabetes and metabolic syndrome.

BACKGROUND

The following discussion refers to a number of publications by author(s)and year of publication, and that due to recent publication datescertain publications are not to be considered as prior art vis-a-vis thepresent invention. Discussion of such publications herein is given formore complete background and is not to be construed as an admission thatsuch publications are prior art for patentability determinationpurposes.

A family of melanocortin receptor types and subtypes have beenidentified, including melanocortin-1 receptors (MC1-R) expressed onnormal human melanocytes and melanoma cells, melanocortin-2 receptors(MC2-R) for ACTH (adrenocorticotropin) expressed in cells of the adrenalgland, melanocortin-3 and melanocortin-4 receptors (MC3-R and MC4-R)expressed primarily in cells in the hypothalamus, mid-brain andbrainstem, and melanocortin-5 receptors (MC5-R), expressed in a widedistribution of peripheral tissues. MC1-R has been suggested to beassociated with hair and skin pigmentation and inflammation, MC2-R isbelieved to mediate steroidogenesis, MC3-R has been suggested to beassociated with energy homeostasis, food intake, and inflammation, MC4-Ris believed to control feeding behavior, energy homeostasis, and sexualfunction (e.g. erectile function), and MC5-R has been suggested to beinvolved in the exocrine gland system.

Significant work has been done in determining the structure ofmelanocortin receptors, including both the nucleic acid sequencesencoding for the receptors and the amino acid sequences constituting thereceptors. MC4-R is a G protein-coupled, 7-transmembrane receptor thatis believed to be expressed primarily in the brain.

MC4-R inactivation has been shown to result in obesity (Hadley, 1999,Ann N.Y. Acad. Sci., 885:1-21). Agouti-related protein (AgRP) is anendogeneous compound that has been suggested to be a MC antagonist or aninverse agonist on MC4-R. The α-melanocyte stimulating hormone (α-MSH)is believed to be the principle endogenous MC4-R agonist.

Also peripherally located MC4-R receptors have been suggested to beinvolved in the control of energy homeostasis, and the role of MC4-Rsignalling in the vagus nerve and its relevance for treatment of obesityand diabetes is discussed by Gautron et al, The Journal of ComparativeNeurology, 518:6-24 (2010).

Peptides specific for MC4-R, and secondarily peptides specific forMC3-R, are believed to be useful in regulation of mammalian energyhomeostasis, including use as agents for attenuating food intake andbody weight gain. MC4-R agonist peptides are believed to be useful fortreating sexual dysfunction, including male erectile dysfunction, andfor decreasing food intake and body weight gain, such as for treatmentof obesity. Such peptides may also be employed for decreasing voluntaryethanol consumption, treatment of drug addictions, and the like. MC4-Ragonist peptides, as well as MC3-R agonist peptides, may further beemployed for treatment of circulatory shock, ischemia, hemorrhagicshock, inflammatory diseases and related diseases, indications,conditions and syndromes. MC4-R antagonist peptides, by contrast, arebelieved to be useful for weight gain aid, such as for use in treatmentof cachexia, sarcopenia, wasting syndrome or disease, and anorexia. Suchpeptides may also be employed for treatment of depression and relateddisorders. (Wikberg et al, Nature Reviews, Drug Discovery, 7, 307,(2008); Adan et al, British J. Pharm., 149, 815-827 (2006); Nogueiras etal, J. Clin., Invest., 117(11): 3475-3488 (2007); Maaser et al, Ann.N.Y. Acad. Sci., 1072, 123-134 (2006); Giuliani et al, British J.Pharm., 150, 595-603 (2007); Balbani, Expert Opin. Ther. Patents, 17(3),287-297 (2007); and Navarro et al, Alcohol. Clin. Exp. Res., 29(6),949-957 (2005)). Melanocortin receptor-specific peptides include cyclicα-MSH analog peptides such asAc-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-NH₂ (SEQ ID NO:1) (See U.S.Pat. Nos. 5,674,839 and 5,576,290) andAc-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-OH (SEQ ID NO:2) (See U.S. Pat.Nos. 6,579,968 and 6,794,489). These and other melanocortinreceptor-specific peptides generally contain the central tetrapeptidesequence of native α-MSH, His⁶-Phe⁷-Arg⁸-Trp⁹ (SEQ ID NO:3), or amimetic or variation thereof, including the substitution of D-Phe forPhe⁷. Other peptides or peptide-like compounds asserted to be specificfor one or more melanocortin receptors are disclosed in U.S. Pat. Nos.5,731,408, 6,054,556, 6,350,430, 6,476,187, 6,600,015, 6,613,874,6,693,165, 6,699,873, 6,887,846, 6,951,916, 7,008,925, and 7,176,279; inU.S. published patent application Publication Nos. 2001/0056179,2002/0143141, 2003/0064921, 2003/0105024, 2003/0212002, 2004/0023859,2005/0130901, 2005/0187164, 2005/0239711, 2006/0105951, 2006/0111281,2006/0293223, 2007/0027091, 2007/0105759, 2007/0123453, 2007/0244054,and 2008/0039387; and in international patent applications nos. WO98/27113, WO 99/21571, WO 00/05263, WO 99/54358, WO 00/35952, WO00/58361, WO 01/30808, WO 01/52880, WO 01/74844, WO 01/85930, WO01/90140, WO 02/18437, WO 02/26774, WO 03/006604, WO 2004/099246, WO2004/046166, WO 2005/000338, WO 2005/000339, WO 2005/000877, WO2005/030797, WO 2005/060985, WO2006/048449, WO 2006/048450, WO2006/048451, WO 2006/048452, WO 2006/097526, WO 2007/008684, WO2007/008704, and WO 2007/009894. Notwithstanding the intense scientificand pharmaceutical interest in melanocortin receptor-specific peptides,evidenced by numerous articles in the scientific literature and numerouspatent applications and issued patents (Nozawa et al, Expert Opin. Ther.Patents 18(4):403-427 (2008); Bednarek et al, Expert Opin. Ther. Patents14(3):327-336 (2004); Todorovic et al, Peptides, 26, 2026-2036 (2005);and Ujjainwalla et al, Current Topics in Med. Chem., 7, 1068-1084(2007)), no melanocortin receptor-specific peptide has been approved asa drug for any therapeutic indication. Indeed, there are no reports ofany melanocortin receptor-specific peptide for any therapeuticindication having advanced past Phase II clinical trials. There remainsa significant and substantial need for melanocortin receptor-specificpeptides for use in pharmaceutical applications. It is against thisbackground that the present invention was made.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide novelcompounds that are useful for the treatment of diseases, disordersand/or conditions responsive to modulation, including activation, ofMC4-R and/or MC3-R, in particular treatment of energy homeostasis andmetabolism related (e.g. diabetes), food intake related and/or energybalance and body weight related diseases, disorders and/or conditions,including obesity, overweight and diseases, disorders and/or conditionsassociated with obesity and/or overweight, such as type 2 diabetes andmetabolic syndrome.

In one aspect, the present invention relates to a cyclic peptide of thestructural Formula (I):

including all enantiomers, stereoisomers or diastereoisomers thereof, ora pharmaceutically acceptable salt of any of the foregoing,

wherein:

-   -   R₁ is —NH—C(═O)— or —C(═O)—NH—;    -   R₂ is —H or —CH₂—, and if R₂ is —CH₂— forms with R₃ a        pyrrolidine ring, the pyrrolidine ring optionally substituted        with —OH;    -   R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

-   -   R_(4a), R_(4b) and R_(4c) are are each independently selected        from hydrogen, halo, (C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl-dihalo,        (C₁-C₁₀)alkyl-trihalo, (C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy,        (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro, nitrile, sulfonamide,        amino, monosubstituted amino, disubstituted amino, hydroxy,        carboxy, or alkoxy-carbonyl, on the proviso that at least one of        R_(4a), R_(4b) and R_(4c) is not hydrogen;    -   R₅ is —OH or —N(R_(6a))(R_(6b));    -   R_(6a) and R_(6b) are each independently H or a C₁ to C₄ linear,        branched or cyclic alkyl chain;    -   R₇ is —H or —C(═O)—NH₂;    -   w is in each instance independently 0 to 5;    -   x is 1 to 5;    -   y is 1 to 5; and    -   z is in each instance independently 1 to 5.

In another aspect, the present invention relates to a cyclic peptide ofFormula (I) is which is of formula (II):

or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention relates to a cyclic peptide ofFormula I, in particular of Formula (II), wherein R₁ is —C(═O)—NH—, x is2 and y is 3.

In another aspect, the present invention relates to a cyclic peptide ofFormula (I), in particular of Formula (II), wherein R₁ is —NH—C(═O)—, xis 3 and y is 2.

In another aspect, the present invention relates to a cyclic peptide ofFormula (I), in particular of Formula (II), wherein R₂ is —H or —CH₂—,and if R₂ is —CH₂— forms with R₃ a pyrrolidine ring, the pyrrolidinering optionally substituted with —OH; and

R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is

wherein w is in each instance independently selected from 0 to 5, and

z is in each instance independently selected from 1 to 5.

In another aspect, the present invention relates to a cyclic peptide ofFormula I, in particular of Formula (II) or (III), wherein R₂ is H, R₃is selected from

In another aspect, the present invention relates to a cyclic peptide ofFormula I, in particular of Formula (II), wherein R₂ is —CH₂— and R₃ is—(CH₂)₂—, R₂ and R₃ together forming an unsubstituted pyrrolidine ring.

In another aspect, the present invention relates to a cyclic peptide ofFormula I, in particular Formula (II), wherein at least one of R_(4a),R_(4b) and R_(4c) is selected from

In another aspect, the present invention relates to a cyclic peptide ofFormula I, in particular Formula (II) or (III), wherein R_(4a) is in the4 position and is —C≡N and R_(4b) and R_(4c) are each H.

In another aspect, the present invention relates to a cyclic peptide ofFormula (I), in particular Formula (II) or (III), wherein R_(4a) is inthe 4 position and is —F and R_(4b) and R_(4c) are each H.

The peptides according to the invention are ligands of one or more ofthe melanocortin receptors, in particular ligands of the MC4-R, moreparticularly agonists (including full and partial agonists) of theMC4-R. The term “ligands” as used herein include peptides binding to theactive site as well as peptides binding to one or more allosteric sitesof any one of said receptors.

Thus, the peptides of the invention can be used as a medicament, inparticular for the treatment of disorders, diseases, or conditionsresponsive to modulation of the MC3-R and/or MC4-R, and in particulardisorders, diseases, or conditions responsive to the activation of theMC4-R. More particularly, the peptides of the invention are believed toattenuate food intake, body weight and/or body weight gain and aretherefore believed to be useful for treatments of energy homeostasis andmetabolism related (e.g. diabetes), food intake related and/or energybalance and body weight related diseases, disorders and/or conditions,including obesity and overweight, and diseases, disorders and/orconditions associated with obesity and/or overweight, such as type 2diabetes and metabolic syndrome, in a patient in need thereof. Thepatient may be a human or non-human animal, in particular a human.

Peptides of the invention may have advantageous properties compared topeptides of the prior art, in particular enhanced potency and/orenhanced selectivity. These advantages may provide for correspondinguseful properties in practice. For example, when used as pharmaceuticalagents, peptides of the present invention may be used at a lower dailyclinical dose, may have longer duration of action, and/or an improvedside effect profile.

In another aspect of the invention, there is provided a method oftreating disorders, diseases, or conditions responsive to modulation ofthe MC4-R and/or MC3-R, such as disorders, diseases, or conditionsresponsive to activation of the MC4-R, in particular energy homeostasisand metabolism related (e.g. diabetes), food intake related and/orenergy balance and body weight related diseases, disorders and/orconditions, including obesity and overweight, and diseases, disordersand/or conditions associated with obesity and/or overweight, such astype 2 diabetes and metabolic syndrome, by administering atherapeutically effective amount of a peptide of the invention to apatient in need thereof.

According to a further aspect of the invention, there is provided amethod of reducing food intake, body weight and/or body weight gain byadministering a pharmacologically effective amount of a peptide of theinvention to an individual, such as a human, in need thereof.

According to a further aspect of the invention, there is provided amethod of preventing body weight regain after weight loss byadministering a pharmacologically effective amount of a peptide of theinvention to an individual, such as a human, in need thereof.

In a further aspect, the invention provides the use of a peptide ofFormula I in the preparation of a medicament for treatment of a disease,disorder and/or condition responsive to activation of the MC4-R, inparticular energy homeostasis and metabolism related (e.g. diabetes),food intake related and/or energy balance and body weight relateddiseases, disorders and/or conditions, including obesity and overweight,and diseases, disorders and/or conditions associated with obesity and/oroverweight, such as type 2 diabetes and metabolic syndrome.

In another aspect, the present invention provides a melanocortinreceptor-specific peptide-based pharmaceutical composition for use intreatment of melanocortin receptor-mediated diseases, indications,conditions and syndromes, in particular diseases, disorders, conditionsand/or syndromes responsive to modulation of MC4-R, such as activationof MC4-R, comprising a peptide of Formula I and a pharmaceuticallyacceptable carrier.

In another aspect, the present invention provides a peptide-basedmelanocortin receptor-specific pharmaceutical composition comprising apeptide of Formula I and a pharmaceutically acceptable carrier, whereinthe peptide is a selective MC4-R ligand, for use in treatment of energyhomeostasis and metabolism related (e.g. diabetes), food intake relatedand/or energy balance and body weight related diseases, disorders and/orconditions, including obesity, overweight and diseases, disorders and/orconditions associated with obesity and/or overweight, such as type 2diabetes and metabolic syndrome.

In another aspect, the present invention provides peptides which arespecific for MC4-R and which are partial or full agonists at MC4-R. Inparticular, the present invention provides peptides which are specificfor MC4-R and which are partial agonists at MC4-R.

In another aspect, the present invention provides a specific MC4-Rcyclic peptide that is effective over a significant dose range.

Other aspects and novel features, and the further scope of applicabilityof the present invention will be set forth in part in the detaileddescription to follow, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The aspects of the invention may be realizedand attained by means of the instrumentalities and combinationsparticularly pointed out in the appended claims.

DETAILED DESCRIPTION OF THE INVENTION 1.0 Definitions

Before proceeding with the description of the invention, certain termsare defined as set forth herein.

In the sequences given for the peptides according to the presentinvention, the amino acid residues have their conventional meaning asgiven in Chapter 2400 of the Manual of Patent Examining Procedure,8^(th) Ed. Thus, “Ala” is alanine, “Asn” is asparagine, “Asp” isaspartic acid, “Arg” is arginine, “Cys” is cysteine, “Gly” is glycine,“Gln” is glutamine, “Glu” is glutamic acid, “His” is histidine, “Ile” isisoleucine, “Leu” is leucine, “Lys” is lysine, “Met” is methionine,“Phe” is phenylalanine, “Pro” is proline, “Ser” is serine, “Thr” isThreonine, “Trp” is tryptophan, “Tyr” is tyrosine, and “Val” is valine,and so on. It is to be understood that “D” isomers are designated by a“D-” before the three letter code or amino acid name, such that forexample D-Phe is D-phenylalanine Amino acid residues not encompassed bythe foregoing have the following definitions:

Abbreviation Common Name Side Chain or Amino Acid Structure Citcitrulline

Dab diaminobutyric acid

Dab(acetyl) 2-amino, 4- acetylamino- butyric acid

Dap diamino- propionic acid

Hyp hydroxyproline

Met(O) methionine sulfoxide

Met(O₂) methionine sulfone

Nle norleucine

Nva norvaline

Orn ornithine

Phe(2-CF₃) 2-trifluoro- methyl phenylalanine

Phe(2-C(═O)—NH₂) 2-carbamoyl- phenylalanine

Phe(2-Me) 2-methyl phenylalanine

Phe(2-CN) 2-cyano phenylalanine

Phe(2-Cl) 2-chloro phenylalanine

Phe(2,4-diCl) 2,4-dichloro phenylalanine

Phe(2,4-diMe) 2,4-dimethyl phenylalanine

Phe(2-F) 2-fluoro phenylalanine

Phe(2-NO₂) 2-nitro phenylalanine

Phe(3-CF₃) 3-trifluoro- methyl phenylalanine

Phe(3-C(═O)—NH₂) 3-carbamoyl- phenylalanine

Phe(3-CN) 3-cyano phenylalanine

Phe(3-Cl) 3-chloro phenylalanine

Phe(3,4-diCl) 3,4-dichloro phenylalanine

Phe(3-F) 3-fluoro phenylalanine

Phe(3,4,5-triF) 3,4,5-trifluoro phenylalanine

Phe(3,4-diF) 3,4-difluoro phenylalanine

Phe(3,5-diF) 3,5-difluoro phenylalanine

Phe(3-Me) 3-methyl phenylalanine

Phe(3-NO₂) 3-nitro phenylalanine

Phe(3,4-diOMe) 3,4-dimethoxy phenylalanine

Phe(4-C(═O)—NH₂) 4=carbamoyl- phenylalanine

Phe(4-Me) 4-methyl phenylalanine

Phe(4-CF₃) 4-trifluoro- methyl phenylalanine

Phe(4-CN) 4-cyano phenylalanine

Phe(4-Cl) 4-chloro phenylalanine

Phe(4-F) 4-fluoro phenylalanine

Phe(4-NH₂) 4-amino phenylalanine

Phe(4-NO₂) 4-nitro phenylalanine

Phe(4-Ph) 4-phenyl phenylalanine

Phe(4-OMe) 4-methoxy phenylalanine

Phe(4-tBu) 4-tert butyl phenylalanine

Ser(Bzl) O-benzyl- serine

Thr(OBzl) O-benzyl- threonine

The term “acyl” includes a group R(C═O)—, where R is an organic group,such as an alkyl, aryl, heteroaryl, carbocyclyl or heterocyclyl. Thus,when reference is made herein to a substituted acyl group, it means thatsaid organic group (R) is substituted. An example is the acetyl groupCH₃—C(═O)—, referred to herein as “Ac”. A peptide or aliphatic moiety is“acylated” when an alkyl or substituted alkyl group as defined above isbonded through one or more carbonyl {—(C═O)—} groups. A peptide is mostusually acylated at the N-terminus.

The term “alkane” includes linear or branched saturated hydrocarbons.Examples of linear alkane groups include methane, ethane, propane, andthe like. Examples of branched or substituted alkane groups includemethylbutane or dimethylbutane, methylpentane, dimethylpentane ortrimethylpentane, and the like. In general, any alkyl group may be asubstitutent of an alkane.

The term “alkene” includes unsaturated hydrocarbons that contain one ormore double carbon-carbon bonds. Examples of such alkene groups includeethylene, propene, and the like.

The term “alkenyl” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one double bond; examplesthereof include ethenyl, 2-propenyl, and the like.

The term “alkyl” includes a straight or branched chain saturatedaliphatic hydrocarbon group. C₁₋₁₀ alkyl means an alkyl having from 1 to10 carbon atoms. Non-limiting examples of such alkyl radicals includemethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl,pentyl, isopentyl, hexyl, isohexyl, and the like.

The term “alkyne” includes a linear monovalent hydrocarbon radical oftwo to six carbon atoms or a branched monovalent hydrocarbon radical ofthree to six carbon atoms containing at least one triple bond; examplesthereof include ethyne, propyne, butyne, and the like.

The term “aryl” includes a monocyclic or bicyclic aromatic hydrocarbonradical of 6 to 12 ring atoms, and optionally substituted independentlywith one or more substituents selected from alkyl, haloalkyl,cycloalkyl, alkoxy, alkythio, halo, nitro, acyl, cyano, amino,monosubstituted amino, disubstituted amino, hydroxy, carboxy, oralkoxy-carbonyl. The term “aryl” also includes bicyclic aromatic ringsystems wherein one ring is aromatic and one ring is non-aromatic(including saturated or partially saturated rings). In bicyclic aromaticring systems, two or more ring carbons are common to two adjoining rings(the rings are “fused rings”). Examples of an aryl group include phenyl,biphenyl, indanyl, naphthyl, 1-naphthyl, and 2-naphthyl, derivativesthereof, and the like.

The term “aralkyl” includes a radical —R^(a)R^(b) where R^(a) is analkylene (a bivalent alkyl) group and R^(b) is an aryl group as definedabove. Examples of aralkyl groups include benzyl, phenylethyl,3-(3-chlorophenyl)-2-methylpentyl, and the like.

The term “aliphatic” includes compounds with hydrocarbon chains, such asfor example alkanes, alkenes, alkynes, and derivatives thereof.

As used herein, the term “amide” includes compounds that have atrivalent nitrogen attached to a carbonyl group, i.e. —C(═O)—NH₂ (i.e.primary amide), —C(═O)—NHR_(c) and —C(═O)—NR_(c)R_(d), wherein each ofR_(c) and R_(d) independently represents an organic group. Whenreference is made herein to a substituted amide group, it means that atleast one of said organic groups (R_(c) and R_(d)) is substituted.Examples of amides include methylamide, ethylamide, propylamide, and thelike.

The term “amine” includes —NH₂ (i.e. an amino group), —NHR_(a) and—NR_(a)R_(b), wherein each of R_(a) and R_(b) independently representsan organic group. When reference is made herein to a substituted aminegroup, it means that at least one of said organic groups (R_(a) andR_(b)) is substituted.

The term “nitrile” includes the functional group —C≡N.

The term “halogen” (or “halo”) is intended to include the halogen atomsfluorine, chlorine, bromine and iodine.

The term “alkyl-halo” includes an alkyl substituted with one halogenatom, such as —CH₂F. The term “alkyl-dihalo” includes an alkylsubstituted with two halogen atoms, such as —CHF₂. The term“alkyl-trihalo” includes an alkyl substituted with three halogen atoms,such as —CF₃.

The term “alkylthio” includes —S-alkyl wherein alkyl is as definedabove. Non-limiting examples of C₁-C₁₀ alkylthio include methylthio,ethylthio, n-propylthio, iso-propylthio, and n-butylthio.

The term “nitro” is intended to include —NO₂.

The term “hydroxy” is intended to include —OH.

The term “alkoxy” includes —O-alkyl wherein alkyl is as defined above.C₁-C₁₀ alkoxy includes an alkyl having from 1 to 10 carbon atoms.Non-limiting examples of C₁-C₁₀ alkoxy include methoxy, ethoxy,n-propyloxy, iso-propyloxy, and 2-methyl-1-propyloxy.

The term “aryloxy” includes —O-aryl wherein aryl is as defined above.

The term “alkoxycarbonyl” includes —C(═O)—O—R, wherein R is an alkyl asdefined above. Non-limiting examples of C₁-C₁₀ alkoxycarbonyl includemethoxycarbonyl, ethoxycarbonyl, isopropoxy-carbonyl andisopentoxycarbonyl.

The term “carboxy” includes —C(═O)OH.

The term “oxo” includes ═O.

The term “sulfonamide” includes a sulfonyl group connected to an aminegroup, i.e. —S(═O)₂NH₂, —S(═O)₂NHR_(a)., and —S(═O)₂NR_(a)R_(b), whereineach of R_(a) and R_(b) independently represents an organic group. Whenreference is made herein to a substituted sulfonamide group, it meansthat at least one of said organic groups (R_(a) and R_(b)) issubstituted.

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) that make up the carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions utilized in the present invention encompassany composition made by admixing an active ingredient and one or morepharmaceutically acceptable carriers.

By a melanocortin receptor “agonist” is meant an endogenous substance,drug substance or compound, including a compound such as the peptides ofthe present invention, which can interact with a melanocortin receptorand initiate a pharmacological response, including but not limited toadenyl cyclase activation, characteristic of the melanocortin receptor.

By “α-MSH” is meant the peptideAc-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (SEQ ID NO:4)and analogs and homologs thereof, including without limitationNDP-α-MSH.

By “NDP-α-MSH” is meant the peptideAc-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (SEQ IDNO:5) and analogs and homologs thereof.

By “EC₅₀” is meant the molar concentration of an agonist, including apartial agonist, which produced 50% of the maximum possible response forthat agonist. By way of example, a test compound which, at aconcentration of 72 nM, produces 50% of the maximum possible responsefor that compound as determined in a cAMP assay in an MC4-R cellexpression system has an EC₅₀ of 72 nM. Unless otherwise specified, themolar concentration associated with an EC₅₀ determination is innanomoles per liter (nM).

By “Ki (nM)” is meant the equilibrium inhibitor dissociation constantrepresenting the molar concentration of a competing compound that bindsto half the binding sites of a receptor at equilibrium in the absence ofradioligand or other competitors. In general, the numeric value of theKi is inversely correlated to the affinity of the compound for thereceptor, such that if the Ki is low, the affinity is high. Ki may bedetermined using the equation of Cheng and Prusoff (Cheng Y., Prusoff W.H., Biochem. Pharmacol. 22: 3099-3108, 1973):

Ki may be expressed in terms of specific receptors (e.g., MC1-R, MC3-R,MC4-R or MC5-R) and specific ligands (e.g. α-MSH or NDP-α-MSH).

By “inhibition” is meant the percent attenuation, or decrease inreceptor binding, in a competitive inhibition assay compared to a knownstandard. Thus, by “inhibition at 1 μM (NDP-α-MSH)” is meant the percentdecrease in binding of NDP-α-MSH by addition of a determined amount ofthe compound to be tested, such as 1 μM of a test compound, such asunder the assay conditions hereafter described. By way of example, atest compound that does not inhibit binding of NDP-α-MSH has a 0%inhibition, and a test compound that completely inhibits binding ofNDP-α-MSH has a 100% inhibition. Typically, as described hereafter, aradio assay is used for competitive inhibition testing, such as withI¹²⁵-labeled NDP-α-MSH, or a lanthanide chelate fluorescent assay, suchas with Eu-NDP-α-MSH. However, other methods of testing competitiveinhibition are known, including use of label or tag systems other thanradioisotopes, and in general any method known in the art for testingcompetitive inhibition may be employed in this invention. It may thus beseen that “inhibition” is one measure to determine whether a testcompound attenuates binding of α-MSH to melanocortin receptors.

By “binding affinity” is meant the ability of a compound or drug to bindto its biological target, expressed herein as Ki (nM).

By “intrinsic activity” is meant the maximal stimulation (functionalactivity) achievable by a compound in a specified melanocortin receptorexpressing cell system, such as the maximal stimulation of adenylylcyclase. The maximal stimulation achieved by α-MSH or NDP-α-MSH isdesignated as an intrinsic activity of 1.0 (or 100%) and a compoundcapable of stimulating half the maximal activity that of α-MSH orNDP-α-MSH is designated as having an intrinsic activity of 0.5 (or 50%).A compound of this invention that under assay conditions describedherein has an intrinsic activity of 0.7 (70%) or higher is classified asan agonist, a compound with intrinsic activity between 0.1 (10%) and 0.7(70%) is classified as a partial agonist, and a compound with intrinsicactivity below 0.1 (10%) is classified as inactive or having nointrinsic activity. In one aspect, the cyclic peptides of the presentinvention may generally be characterized as a partial agonist at MC4-Rwith respect to α-MSH or NDP-α-MSH.

In general, “functional activity” is a measure of the signaling of areceptor, or measure of a change in receptor-associated signaling, suchas a melanocortin receptor, and in particular MC4-R or hMC4-R, uponactivation by a compound. Melanocortin receptors initiate signaltransduction through activation of heterotrimeric G proteins. In oneaspect, melanocortin receptors signal through Gα_(S), which catalyzesproduction of cAMP by adenylyl cyclase. Thus determination ofstimulation of adenylyl cyclase, such as determination of maximalstimulation of adenylyl cyclase, is one measure of functional activity,and is the primary measure exemplified herein. However, it is to beunderstood that alternative measures of functional activity may beemployed in the practice of this invention, and are specificallycontemplated and included within the scope of this invention. Thus, inone example intracellular free calcium may be measured, such as reportedby and using the methods disclosed in Mountjoy K. G. et al.,Melanocortin receptor-medicated mobilization of intracellular freecalcium in HEK293 cells. Physiol Genomics 5:11-19, 2001, or Kassack M.U. et al., Functional screening of G protein-coupled receptors bymeasuring intracellular calcium with a fluorescence microplate reader.Biomol Screening 7:233-246, 2002. It is also possible to measureactivation by measurement of the production of inositol triphosphate ordiacylglycerol from phosphatidylinositol 4,5-biphosphate, such as by useof radioassays. Yet another measure of functional activity is receptorinternalization, resulting from activation of regulatory pathways, suchas using the methods disclosed in Nickolls S. A. et al., Functionalselectivity of melanocortin 4 receptor peptide and nonpeptide agonists:evidence for ligand specific conformational states. J Pharm ExperTherapeutics 313:1281-1288, 2005. Yet another measure of functionalactivity is the exchange, and exchange rate, of nucleotides associatedwith activation of a G protein receptor, such as the exchange of GDP(guanosine diphosphate) for GTP (guanosine triphosphase) on the Gprotein α subunit, which may be measured by any number of means,including a radioassay using guanosine 5′-(γ-[³⁵S]thio)-triphosphate, asdisclosed in Manning D. R., Measures of efficacy using G proteins asendpoints: differential engagement of G proteins through singlereceptors. Mol Pharmacol 62:451-452, 2002. Various gene-based assayshave been developed for measuring activation of G-coupled proteins, suchas those disclosed in Chen W. et al., A colorimetric assay frommeasuring activation of Gs- and Gq-coupled signaling pathways. AnalBiochem 226:349-354, 1995; Kent T. C. et al., Development of a genericdual-reporter gene assay for screening G-protein-coupled receptors.Biomol Screening, 5:437-446, 2005; or Kotarsky K. et al., Improvedreceptor gene assays used to identify ligands acting on orphanseven-transmembrane receptors. Pharmacology & Toxicology 93:249-258,2003. The colorimetric assay of Chen et al. has been adapted for use inmeasuring melanocortin receptor activation, as disclosed in Hruby V. J.et al., Cyclic lactam α-melanocortin analogues of Ac-Nle⁴-cyclo[Asp⁵,D-Phe⁷, Lys¹⁰] α-melanocyte-stimulating hormone-(4-10)-NH₂ with bulkyaromatic amino acids at position 7 shows high antagonist potency andselectivity at specific melanocortin receptors. J Med Chem 38:3454-3461,1995. In general, functional activity may be measured by any method,including methods of determining activation and/or signaling of aG-coupled receptor, and further including methods which may be hereafterdeveloped or reported. The terms “treat,” “treating” and “treatment,” asused herein, contemplate an action that occurs while a patient issuffering from the specified disease, disorder and/or condition, whichreduces the severity of the disease, disorder and/or condition.Moreover, the terms “treat,” “treating” and “treatment,” as used hereinare intended to embrace therapeutic (curative), prophylactic(preventing), controlling and palliative treatment of the indicateddiseases, disorders and/or conditions.

As used herein, the term “pharmacologically effective amount” (including“therapeutically effective amount”) means an amount of a peptideaccording to the invention that is sufficient to induce a desiredtherapeutic or biological effect.

As used herein, the term “therapeutically effective amount” means theamount of a peptide of the invention that will elicit a biological ormedical response in the mammal that is being treated by a medical doctoror other clinician.

As used herein, the term “prophylactically effective” or “preventive”means the amount of a peptide of the invention that will prevent orinhibit affliction or mitigate affliction of a mammal with a medicalcondition that a medical doctor or other clinician is trying to prevent,inhibit, or mitigate before a patient begins to suffer from thespecified disease or disorder.

The term “diabetes” includes Type 1 Diabetes, which is insulin-dependentdiabetes mellitus as diagnosed according to criteria published in theReport of the Expert Committee on the Diagnosis and Classification ofDiabetes Mellitus (Diabetes Care, Vol. 24, Supp. 1, January 2001)whereby fasting plasma glucose level is greater than or equal to 126milligrams per deciliter and for which the primary cause is pancreaticbeta cell destruction, Type 2 Diabetes, which is non-insulin-dependentdiabetes mellitus as diagnosed according to criteria published in theReport of the Expert Committee on the Diagnosis and Classification ofDiabetes Mellitus whereby fasting plasma glucose level is greater thanor equal to 126 milligrams per deciliter, and latent autoimmune diabetesmellitus of adults (LADA).

The term “metabolic syndrome” refers to metabolic disorders,particularly glucose and lipid regulatory disorders, including insulinresistance and defective secretion of insulin by pancreatic beta cells,and may further include conditions and states such as abdominal obesity,dyslipidemia, hypertension, glucose intolerance or a prothrombiticstate, and which may further result in disorders such as hyperlipidemia,obesity, diabetes, insulin resistance, glucose intolerance,hyperglycemia, and hypertension.

2.0 Clinical Indications and Utility

The compositions and methods disclosed herein can be used for bothmedical applications and animal husbandry or veterinary applications.Typically, the methods are used in humans, but may also be used in othermammals. The term “patient” is intended to denote a mammalianindividual, and is so used throughout the specification and in theclaims. The primary applications of the present invention involve humanpatients, but the present invention may be applied to laboratory, farm,zoo, wildlife, pet, sport or other animals. Clinical indications andspecific utilities include the following:

2.1 Obesity and Related Metabolic Syndrome.

Peptides of Formula (I), and in particular Formula (II) or (III), havebeen found to be ligands of the MC4 receptor. In particular, peptides ofFormula (I) are believed to be useful in treating diseases, disordersand/or conditions responsive to modulation of the MC4-R function, moreparticularly activation of the MC4-R, i.e. diseases, disorders and/orconditions which would benefit from agonism (including full or partialagonism) at the MC4-R, including energy homeostasis and metabolismrelated (such as diabetes, in particular type 2 diabetes; dyslipidemia;fatty liver; hypercholesterolemia; hypertriglyceridemia;hyperuricacidemia; impaired glucose tolerance; impaired fasting glucos;insulin resistance syndrome; and metabolic syndrome), food intakerelated (such as hyperphagia; binge eating; bulimia; and compulsiveeating) and/or energy balance and body weight related diseases,disorders and/or conditions, more particularly such diseases, disordersand conditions characterized by excess body weight and/or excess foodintake.

Peptides of Formula (I), and in particular Formula (II) or (III), areparticularly believed to be useful for treatment of body weight relateddiseases, disorders and/or conditions characterized by excess bodyweight, including obesity and overweight (by promotion of weight loss,maintenance of weight loss, and/or prevention of weight gain, includingmedication-induced weight gain or weight gain subsequent to cessation ofsmoking), and diseases, disorders and/or conditions associated withobesity and/or overweight, such as insulin resistance; impaired glucosetolerance; type 2 diabetes; metabolic syndrome; dyslipidemia (includinghyperlipidemia); hypertension; heart disorders (e.g. coronary heartdisease, myocardial infarction); cardiovascular disorders; non-alcoholicfatty liver disease (including non-alcoholic steatohepatitis); jointdisorders (including secondary osteoarthritis); gastroesophageal reflux;sleep apnea; atherosclerosis; stroke; macro and micro vascular diseases;steatosis (e.g. in the liver); gall stones; and gallbladder disorders.

Peptides of Formula (I), and in particular Formula (II) or (III), areparticularly believed to be useful for treatment of obesity and type 2diabetes, more specifically obesity.

It will be understood that there are medically accepted definitions ofobesity and overweight. A patient may be identified by, for example,measuring body mass index (BMI), which is calculated by dividing weightin kilograms by height in meters squared, and comparing the result withthe definitions. The recommended classifications for BMI in humans,adopted by the Expert Panel on the Identification, Evaluation andTreatment of Overweight and Obesity in Adults, and endorsed by leadingorganizations of health professionals, are as follows: underweight <18.5kg/m², normal weight 18.5-24.9 kg/m², overweight 25-29.9 kg/m₂, obesity(class 1) 30-34.9 kg/m², obesity (class 2) 35-39.9 kg/m², extremeobesity (class 3)≧40 kg/m² (Practical Guide to the Identification,Evaluation, and Treatment of Overweight and Obesity in Adults, The NorthAmerican Association for the Study of Obesity (NAASO) and the NationalHeart, Lung and Blood Institute (NHLBI) 2000). Modifications of thisclassification may be used for specific ethnic groups and for children.Another alternative for assessing overweight and obesity is by measuringwaist circumference. There are several proposed classifications anddifferences in the cutoffs based on ethnic group. For instance,according to the classification from the International DiabetesFederation, men having waist circumferences above 94 cm (cut off foreuropids) and women having waist circumferences above 80 cm (cut off foreuropids) are at higher risk of diabetes, dyslipidemia, hypertension andcardiovascular diseases because of excess abdominal fat. Anotherclassification is based on the recommendation from the Adult TreatmentPanel III where the recommended cut-offs are 102 cm for men and 88 cmfor women. However, the peptides of Formula (I) may also be used forreduction of self-diagnosed overweight and for decreasing the risk ofbecoming obese due to life style, genetic considerations, heredityand/or other factors.

It is believed that peptides of Formula (I), and in particular Formula(II) or (III), upon administration to an animal, including man, willreduce food intake, body weight and/or body weight gain in that animal.

Without being bound by any theory, it is believed that peptides ofFormula (I), and in particular Formula (II) or (III), act by modulatingappetite and/or satiety, increasing metabolic rate, reducing intake ofand/or craving for fat and/or carbohydrates.

Without being bound by any theory, it is also believed that peptides ofFormula (I), and in particular Formula (II) or (III), act by enhancingglucose tolerance and/or decreasing insulin resistance. It is thereforebelieved that peptides of Formula (I) can be useful also for treatmentof type 2 diabetes in underweight and normal weight individuals as wellas in overweight and obese individuals.

Peptides of the invention might also be useful for (i) prevention oforgan or tissue damage caused by hypoperfusion due to vessel occlusion(e.g. caused by thrombosis), haemorrhage, trauma, surgery, haemorrhagicshock, cardiogenic shock, toxic shock or septic shock or (iii) treatmentof male and female sexual dysfunctions, such as male erectiledysfunction or female sexual arousal dysfunction.

According to a further aspect of the invention, there is provided apeptide of Formula (I), and in particular Formula (II) or (III), aspreviously defined for use as a medicament.

In another aspect, the invention provides the use of a peptide ofFormula (I), and in particular Formula (II) or (III), for treatment ofdiseases, disorders and/or conditions responsive to modulation of theMC4-R, such as diseases, disorders and/or conditions responsive toactivation of the MC4-R, in particular energy homeostasis and metabolismrelated (e.g. diabetes), food intake related and/or energy balance andbody weight related diseases, disorders and/or conditions, includingobesity, overweight and diseases, disorders and/or conditions associatedwith obesity and/or overweight, such as type 2 diabetes and metabolicsyndrome.

In a further aspect, the invention provides the use of a peptide ofFormula (I), and in particular Formula (II) or (III), in the preparationof a medicament for treatment of diseases, disorders and/or conditionsresponsive to modulation of the MC4-R, such as activation of the MC4-R,in particular energy homeostasis and metabolism related (e.g. diabetes),food intake related and/or energy balance and body weight relateddiseases, disorders and/or conditions, including obesity, overweight anddiseases, disorders and/or conditions associated with obesity and/oroverweight, such as type 2 diabetes and metabolic syndrome.

Peptides of the invention are advantageously more selective (i.e. higheraffinity and/or higher specificity) for MC4-R and/or MC3-R than forMC1-R, MC2-R and MC5-R, in particular MC1-R. In particular, peptides ofthe invention are advantageously more selective for MC4-R than for anyof MC3-R and MC1-R. Peptides of the invention are suitably at least3-fold, in particular 10-fold, more particularly 30-fold, more selectivefor MC4-R than for any of MC1-R, MC2-R, MC3-R and MC5-R. Some peptidesof the invention are even more than 100-fold, such as even about150-fold, more selective for MC4-R than MC1-R as determined in thereceptor binding assay described in 7.1. It is noted that theselectivity profile may affect in vivo safety and side effects obtainedupon administration of the peptides.

Upon treatment of energy homeostasis and metabolism related, food intakerelated and/or energy balance and body weight related diseases,disorders and/or conditions, it is desirable to reduce or eliminateunwanted side-effects that may result from MC4-R activation, such assexual side-effects, including penile erection, and blood pressureeffects.

Peptides of the invention are believed to be useful for treatment ofenergy homeostasis and metabolism related (e.g. diabetes), food intakerelated and/or energy balance and body weight related diseases,disorders and/or conditions, including obesity, overweight and diseases,disorders and/or conditions associated with obesity and/or overweight,such as type 2 diabetes and metabolic syndrome, without causingsubstantial adverse cardiovascular effects, including a substantialincrease in blood pressure.

Peptides of the invention are believed to be useful for treatment ofenergy homeostasis and metabolism related (e.g. diabetes), food intakerelated and/or energy balance and body weight related diseases,disorders and/or conditions, including obesity, overweight and diseases,disorders and/or conditions associated with obesity and/or overweight,such as type 2 diabetes and metabolic syndrome, without causingsubstantial unwanted sexual effects resulting from MC4-R activation,such as penile erection.

It is believed that peptides of Formula I, and in particular Formula(II) or (III), possess a satisfactory pharmacological profile andpromising biopharmaceutical properties, such as toxicological profile,metabolism and pharmacokinetic properties, solubility, and permeability.It will be understood that determination of appropriatebiopharmaceutical properties is within the knowledge of a person skilledin the art.

3.0 Combination Therapy for Certain Indications

The peptides, compositions and methods of the present invention may beused for treatment of any of the foregoing diseases, indications,conditions or syndromes, or any disease, indication, condition orsyndrome which is melanocortin receptor mediated, by administration incombination with one or more other pharmaceutically active compounds.Such combination administration may be by means of a single dosage formwhich includes both a peptide of the present invention and one moreother pharmaceutically active compound, such single dosage formincluding a tablet, capsule, spray, inhalation powder, injectable liquidor the like. Alternatively, combination administration may be by meansof administration of two different dosage forms, with one dosage formcontaining a peptide of the present invention, and the other dosage formincluding another pharmaceutically active compound. In this instance,the dosage forms may be the same or different. Without meaning to limitcombination therapies, the following exemplifies certain combinationtherapies which may be employed.

3.1 Combination Therapy for Obesity and Related Metabolic Syndrome.

One or more peptides of the invention may be combined with one or moreother pharmacologically active agent(s) that is (are) useful in thetreatment of various weight and feeding-related disorders, such asobesity and/or overweight, in particular other anti-obesity drugs thataffect energy expenditure, glycolysis, gluconeogenesis, glucogenolysis,lipolysis, lipogenesis, fat absorption, fat storage, fat excretion,hunger and/or satiety and/or craving mechanisms, appetite/motivation,food intake, or gastrointestinal motility. Drugs that reduce energyintake include, in part, various pharmacological agents, referred to asanorectic drugs, which are used as adjuncts to behavioral therapy inweight reduction programs.

Generally, a total dosage of the below-described obesity control agentsor medications, when used in combination with one or more peptides ofthe present invention can range from 0.1 to 3,000 mg/day, preferablyfrom about 1 to 1,000 mg/day and more preferably from about 1 to 200mg/day in single or 2-4 divided doses. The exact dose, however, isdetermined by the attending clinician and is dependent on such factorsas the potency of the compound administered, the age, weight, conditionand response of the patient.

One or more peptides of the invention may be combined with one or moreother pharmacologically active agent(s) that is (are) useful in thetreatment of diabetes, such as other anti-diabetic drugs.

One or more peptides of the invention may in addition or alternativelyfurther be combined with one or more other pharmacologically activeagent(s) that is (are) useful in the treatment of diseases, disordersand/or conditions associated with obesity and/or overweight, such asinsulin resistance; impaired glucose tolerance; type 2 diabetes;metabolic syndrome; dyslipidemia (including hyperlipidemia);hypertension; heart disorders (e.g. coronary heart disease, myocardialinfarction); cardiovascular disorders; non-alcoholic fatty liver disease(including non-alcoholic steatohepatitis); joint disorders (includingsecondary osteoarthritis); gastroesophageal reflux; sleep apnea;atherosclerosis; stroke; macro and micro vascular diseases; steatosis(e.g. in the liver); gall stones; and gallbladder disorders.

According to a further aspect of the invention there is provided acombination treatment comprising the administration of apharmacologically effective amount of a peptide according to theinvention, or a pharmaceutically acceptable salt thereof, optionallytogether with a pharmaceutically acceptable diluent or carrier, with thesimultaneous, sequential or separate administration one or more of thefollowing agents selected from:

-   -   insulin and insulin analogues;    -   insulin secretagogues, including sulphonylureas (e.g. glipizide)        and prandial glucose regulators (sometimes called “short-acting        secretagogues”), such as meglitinides (e.g. repaglinide and        nateglinide);    -   agents that improve incretin action, for example dipeptidyl        peptidase IV (DPP-4) inhibitors (e.g. vildagliptin, saxagliptin,        and sitagliptin), and glucagon-like peptide-1 (GLP-1) agonists        (e.g. exenatide);    -   insulin sensitising agents including peroxisome proliferator        activated receptor gamma (PPARγ) agonists, such as        thiazolidinediones (e.g. pioglitazone and rosiglitazone), and        agents with any combination of PPAR alpha, gamma and delta        activity;    -   agents that modulate hepatic glucose balance, for example        biguanides (e.g. metformin), fructose 1,6-bisphosphatase        inhibitors, glycogen phopsphorylase inhibitors, glycogen        synthase kinase inhibitors, and glucokinase activators;    -   agents designed to reduce/slow the absorption of glucose from        the intestine, such as alpha-glucosidase inhibitors (e.g.        miglitol and acarbose);    -   agents which antagonise the actions of or reduce secretion of        glucagon, such as amylin analogues (e.g. pramlintide);    -   agents that prevent the reabsorption of glucose by the kidney,        such as sodium-dependent glucose transporter 2 (SGLT-2)        inhibitors (e.g. dapagliflozin);    -   agents designed to treat the complications of prolonged        hyperglycaemia, such as aldose reductase inhibitors (e.g.        epalrestat and ranirestat); and agents used to treat        complications related to micro-angiopathies;    -   anti-dyslipidemia agents, such as HMG-CoA reductase inhibitors        (statins, e.g. rosuvastatin) and other cholesterol-lowering        agents; PPARα agonists (fibrates, e.g. gemfibrozil and        fenofibrate); bile acid sequestrants (e.g. cholestyramine);    -   cholesterol absorption inhibitors (e.g. plant sterols (i.e.        phytosterols), synthetic inhibitors); cholesteryl ester transfer        protein (CETP) inhibitors; inhibitors of the ileal bile acid        transport system (IBAT inhibitors); bile acid binding resins;        nicotinic acid (niacin) and analogues thereof; anti-oxidants,        such as probucol; and omega-3 fatty acids;    -   antihypertensive agents, including adrenergic receptor        antagonists, such as beta blockers (e.g. atenolol), alpha        blockers (e.g. doxazosin), and mixed alpha/beta blockers (e.g.        labetalol); adrenergic receptor agonists, including alpha-2        agonists (e.g. clonidine); angiotensin converting enzyme (ACE)        inhibitors (e.g. lisinopril), calcium channel blockers, such as        dihydropyridines (e.g. nifedipine), phenylalkylamines (e.g.        verapamil), and benzothiazepines (e.g. diltiazem); angiotensin        II receptor antagonists (e.g. candesartan); aldosterone receptor        antagonists (e.g. eplerenone); centrally acting adrenergic        drugs, such as central alpha agonists (e.g. clonidine); and        diuretic agents (e.g. furosemide);    -   haemostasis modulators, including antithrombotics, such as        activators of fibrinolysis; thrombin antagonists; factor VIIa        inhibitors; anticoagulants, such as vitamin K antagonists (e.g.        warfarin), heparin and low molecular weight analogues thereof,        factor Xa inhibitors, and direct thrombin inhibitors (e.g.        argatroban); antiplatelet agents, such as cyclooxygenase        inhibitors (e.g. aspirin), adenosine diphosphate (ADP) receptor        inhibitors (e.g. clopidogrel), phosphodiesterase inhibitors        (e.g. cilostazol), glycoprotein IIB/IIA inhibitors (e.g.        tirofiban), and adenosine reuptake inhibitors (e.g.        dipyridamole);    -   anti-obesity agents, such as appetite suppressant (e.g.        ephedrine), including noradrenergic agents (e.g. phentermine)        and serotonergic agents (e.g. sibutramine), pancreatic lipase        inhibitors (e.g. orlistat), microsomal transfer protein (MTP)        modulators, diacyl glycerolacyltransferase (DGAT) inhibitors,        and cannabinoid (CB1) receptor antagonists (e.g. rimonabant);    -   feeding behavior modifying agents, such as orexin receptor        modulators and melanin-concentrating hormone (MCH) modulators;    -   glucagon like peptide-1 (GLP-1) receptor modulators;    -   neuropeptideY (NPY)/NPY receptor modulators;    -   pyruvate dehydrogenase kinase (PDK) modulators;    -   serotonin receptor modulators;    -   leptin/leptin receptor modulators;    -   ghrelin/ghrelin receptor modulators; or    -   monoamine transmission-modulating agents, such as selective        serotonin reuptake inhibitors (SSRI) (e.g. fluoxetine),        noradrenaline reuptake inhibitors (NARI),        noradrenaline-serotonin reuptake inhibitors (SNRI), triple        monoamine reuptake blockers (e.g. tesofensine), and monoamine        oxidase inhibitors (MAOI) (e.g. toloxatone and amiflamine),        or a pharmaceutically acceptable salt, solvate, solvate of such        a salt or a prodrug thereof, optionally together with a        pharmaceutically acceptable carrier to a mammal, such as man, in        need of such therapeutic treatment.

According to an additional further aspect of the present invention thereis provided a combination treatment comprising the administration of apharmacologically effective amount of a compound according to theinvention, or a pharmaceutically acceptable salt thereof, optionallytogether with a pharmaceutically acceptable carrier, with thesimultaneous, sequential or separate administration of very low caloriediets (VLCD) or low-calorie diets (LCD).

4.0 Methods of Making

In general, the peptides of the present invention may be synthesized bysolid-phase synthesis and purified according to methods known in theart. Any of a number of well-known procedures utilizing a variety ofresins and reagents may be used to prepare the peptides of the presentinvention.

The cyclic peptides of the present invention may be readily synthesizedby known conventional procedures for the formation of a peptide linkagebetween amino acids. Such conventional procedures include, for example,any solution phase procedure permitting a condensation between the freealpha amino group of an amino acid or residue thereof having itscarboxyl group and other reactive groups protected and the free primarycarboxyl group of another amino acid or residue thereof having its aminogroup or other reactive groups protected. In a preferred conventionalprocedure, the cyclic peptides of the present invention may besynthesized by solid-phase synthesis and purified according to methodsknown in the art. Any of a number of well-known procedures utilizing avariety of resins and reagents may be used to prepare the peptides ofthe present invention.

The process for synthesizing the cyclic peptides may be carried out by aprocedure whereby each amino acid in the desired sequence is added oneat a time in succession to another amino acid or residue thereof or by aprocedure whereby peptide fragments with the desired amino acid sequenceare first synthesized conventionally and then condensed to provide thedesired peptide. The resulting peptide is then cyclized to yield acyclic peptide of the invention.

Solid phase peptide synthesis methods are well known and practiced inthe art. In such methods the synthesis of peptides of the invention canbe carried out by sequentially incorporating the desired amino acidresidues one at a time into the growing peptide chain according to thegeneral principles of solid phase methods. These methods are disclosedin numerous references, including Merrifield, R. B., Solid phasesynthesis (Nobel lecture). Angew Chem 24:799-810 (1985) and Barany etal., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E.and Meienhofer, J., Eds. Academic Press 1-284 (1980).

In chemical syntheses of peptides, reactive side chain groups of thevarious amino acid residues are protected with suitable protectinggroups, which prevent a chemical reaction from occurring at that siteuntil the protecting group is removed. Also common is the protection ofthe alpha amino group of an amino acid residue or fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the alpha amino protecting group to allow a subsequent reaction totake place at that site. Specific protecting groups have been disclosedand are known in solid phase synthesis methods and solution phasesynthesis methods.

Alpha amino groups may be protected by a suitable protecting group,including a urethane-type protecting group, such as benzyloxycarbonyl(Z) and substituted benzyloxycarbonyl, such asp-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl,9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz)and aliphatic urethane-type protecting groups, such ast-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl,isopropoxycarbonyl, and allyloxycarbonyl (Alloc). Fmoc are preferred foralpha amino protection.

Guanidino groups may be protected by a suitable protecting group, suchas nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc),adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) andBoc. Pbf and Pmc are preferred protecting groups for Arg.

The peptides of the invention described herein were prepared using solidphase synthesis, such as by means of a Symphony Multiplex PeptideSynthesizer (Rainin Instrument Company/Protein Technologies Inc)automated peptide synthesizer, using programming modules as provided bythe manufacturer and following the protocols set forth in themanufacturer's manual.

Solid phase synthesis is commenced from the C-terminal end of thepeptide by coupling a protected alpha amino acid to a suitable resin.Such starting material is prepared by attaching an alpha amino-protectedamino acid by an amide linkage to a 4-(2′,4′-dimethoxylphenyl-aminomethyl-phenoxy (Rink Amide) resin, a4-(2′,4′-dimethoxylphenyl-aminomethyl)-phenoxyacetamido-norleucyl-MBHAresin, an amino-xanthen-3-yloxy-merifiel resin (Sieber Amide) resin, orby an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin, a2-chlorotrityl chloride resin or by other means well known in the art.Fmoc-Linker-BHA resin supports are commercially available and generallyused when feasible. The resins are carried through repetitive cycles asnecessary to add amino acids sequentially. The alpha amino Fmocprotecting groups are removed under basic conditions. Piperidine,piperazine, diethylamine, or morpholine (20-40% v/v) inN,N-dimethylformamide (DMF) may be used for this purpose.

Following removal of the alpha amino protecting group, the subsequentprotected amino acids are coupled stepwise in the desired order toobtain an intermediate, protected peptide-resin. The activating reagentsused for coupling of the amino acids in the solid phase synthesis of thepeptides are well known in the art. After the peptide is synthesized, ifdesired, the orthogonally protected side chain protecting groups may beremoved using methods well known in the art for further derivatizationof the peptide.

Typically, orthogonal protecting groups are used as appropriate. Forexample, the peptides of the invention contain multiple amino acids withan amino group-containing side chain. In one aspect, an Allyl-Allocprotection scheme is employed with the amino acids forming a lactambridge through their side chains, and orthogonal protecting groups,cleavable under different reactive conditions, use for other amino acidswith amino group-containing side chains. Thus, for example,Fmoc-Orn(Alloc)-OH and Fmoc-Glu(OAll)-OH amino acids (Glu(OAll) refersto glutamic acid 5-allyl ester) can be employed for the positionsforming a lactam bridge upon cyclization, while other amino acids withamino group-containing side chains have a different and orthogonalprotecting group, such as with Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Pbf)-OH,Fmoc-Dab(Pbf)-OH or the like. Other protecting groups may be similarlyemployed; by way of example and not limitation, Mtt/OPp(4-methyltrityl/2-phenylisopropyl) can be employed with the side chainsforming a lactam bridge upon cyclization, with orthogonal protectinggroups being utilized for other positions that are not cleavable usingconditions suitable for cleavage of Mtt/OPp.

Reactive groups in a peptide can be selectively modified, either duringsolid phase synthesis or after removal from the resin. For example,peptides can be modified to obtain N-terminus modifications, such asacetylation, while on resin, or may be removed from the resin by use ofa cleaving reagent and then modified. Similarly, methods for modifyingside chains of amino acids are well known to those skilled in the art ofpeptide synthesis. The choice of modifications made to reactive groupspresent on the peptide will be determined, in part, by thecharacteristics that are desired in the peptide.

In the peptides of the present invention, in one embodiment theN-terminus group is modified by introduction of an N-acetyl group. Inone aspect, a method is employed wherein after removal of the protectinggroup at the N-terminal, the resin-bound peptide is reacted with aceticanhydride in DMF in the presence of an organic base, such aspyridine.Other methods of N-terminus acetylation are known in the art, includingsolution phase acetylation, and may be employed.

The peptide can, in one embodiment, be cyclized prior to cleavage fromthe peptide resin. For cyclization through reactive side chain moieties,the desired side chains are deprotected, and the peptide suspended in asuitable solvent and a cyclic coupling agent added. Suitable solventsinclude, for example DMF, dichloromethane (DCM) or1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling reagents include,for example, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU),benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP),benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate(PyBOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TATU),2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TPTU) or N,N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazole(DCCI/HOBt). Coupling is conventionally initiated by use of a suitablebase, such as N,N-diisopropylethylamine (DIPEA), sym-collidine orN-methylmorpholine (NMM).

The cyclized peptides can then be cleaved from solid phase, using anysuitable reagent, such as ethylamine in DCM or various combinations ofagents, such as trifluoroacetic acid (TFA), tri-isopropylsilane (TIS),dimethoxybenezene (DMB), water and the like. The resulting crude peptideis dried and remaining amino acid side chain protecting groups, if any,are cleaved using any suitable reagent, such as TFA in the presence ofwater, TIS, 2-mercaptopethane (ME), and/or 1,2-ethanedithiol (EDT). Thefinal product is precipitated by adding cold ether and collected byfiltration. Final purification is by reverse phase high performanceliquid chromatography (RP-HPLC), using a suitable column, such as a C₁₈column, or other methods of separation or purification, such as methodsbased on the size or charge of the peptide, can also be employed. Oncepurified, the peptide can be characterized by any number of methods,such as high performance liquid chromatography (HPLC), amino acidanalysis, mass spectrometry, and the like.

For peptides of the present invention which have a C-terminussubstituted amide derivative or N-alkyl group, synthesis may proceed bysolid phase synthesis commenced from the C-terminal end of the peptideby coupling a protected alpha amino acid to a suitable resin. Suchmethods for preparing substituted amide derivatives on solid-phase havebeen described in the art. See, for example, Barn D. R. et al.,Synthesis of an array of amides by aluminum chloride assisted cleavageon resin bound esters. Tetrahedron Letters, 37:3213-3216 (1996); DeGradoW. F. and Kaiser E. T., Solid-phase synthesis of protected peptides on apolymer bound oxime: Preparation of segments comprising the sequences ofa cytotoxic 26-peptide analogue. J. Org. Chem., 47:3258-3261 (1982).Such a starting material can be prepared by attaching an alphaamino-protected amino acid by an ester linkage to a p-benzyloxybenzylalcohol (Wang) resin or an by amide linkage to a4-(2′,4′-dimethoxylphenyl-aminomethyl-phenoxy (Rink Amide) resin by wellknown means. The peptide chain is grown with the desired sequence ofamino acids. Before cleavage, the peptide is cyclized on the solidphase. Peptides employing a p-benzyloxybenzyl alcohol (Wang) resin maybe cleaved from resin by aluminum chloride in DCM, and peptidesemploying a Rink Amide resin may be cleaved by mixture of TFA, TIS andwater.

While synthesis has been described primarily with reference to solidphase Fmoc chemistry, it is to be understood that other chemistries andsynthetic methods may be employed to make the cyclic peptides of theinvention, such as by way of example and not limitation, methodsemploying Boc chemistry, solution chemistry, and other chemistries andsynthetic methods.

5.1 Salt Form of Cyclic Peptides of the Present Invention.

It shall be understood that as used herein all references to peptidesaccording to the invention, including a specific chemical formula orname, are intended to include all pharmaceutically acceptable salts,solvates, hydrates, polymorphs, prodrugs, metabolites, stereoisomers,and tautomeric isomers thereof.

Cyclic peptides of the present invention may be in the form of anypharmaceutically acceptable salt. The term “pharmaceutically acceptablesalts” refers to salts prepared from pharmaceutically acceptablenon-toxic bases or acids including inorganic or organic bases andinorganic or organic acids (see “Handbook of Pharmaceutical Salts:Properties, Selection and Use”, P. H. Stahl, P. G. Wermuth, IUPAC,Wiley-VCH, 2002). Salts derived from inorganic bases include aluminum,ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganicsalts, manganous, potassium, sodium, zinc, and the like. Particularlypreferred are the ammonium, calcium, lithium, magnesium, potassium, andsodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines, and basic ion exchange resins, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like. When cyclic peptide of thepresent invention is basic, acid addition salts may be prepared frompharmaceutically acceptable non-toxic acids, including inorganic andorganic acids. Such acids include acetic, benzenesulfonic, benzoic,camphorsulfonic, carboxylic, citric, ethanesulfonic, formic, fumaric,gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic,maleic, malic, mandelic, methanesulfonic, malonic, mucic, nitric,pamoic, pantothenic, phosphoric, propionic, succinic, sulfuric,tartaric, p-toluenesulfonic acid, trifluoroacetic acid, and the like.Acid addition salts of peptides of the present invention are prepared ina suitable solvent for the peptide and an excess of an acid, such ashydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic(TFA), citric, tartaric, maleic, succinic or methanesulfonic acid. Theacetate, ammonium acetate and TFA acid salt forms are especially useful.

Where peptides of the present invention include an acidic moiety,suitable pharmaceutically acceptable salts may include alkali metalsalts, such as sodium or potassium salts, or alkaline earth metal salts,such as calcium or magnesium salts. It is also to be understood thatcertain peptides of the Formula (I) can exist in solvated forms,including solvates of the free peptide or solvates of a salt of thecompound, as well as unsolvated forms. The term “solvate” is used hereinto describe a molecular complex comprising the compound of the inventionand one or more pharmaceutically acceptable solvent molecules, forexample, ethanol. The term “hydrate” is employed when said solvent iswater. It is to be understood that all polymorphs, including mixtures ofdifferent polymorphs, are included within the scope of the claimedpeptides.

5.2 Pharmaceutical Compositions.

The invention provides a pharmaceutical composition that includes one ormore cyclic peptides of the present invention and a pharmaceuticallyacceptable carrier. When formulated with a pharmaceutically acceptablecarrier, the compound of the invention may be present in thepharmaceutical composition in a concentration from 0.1 to 99.5%, such asfrom 0.5 to 95%, by weight of the total composition. The choice ofcarrier is within the knowledge of a person skilled in the art anddepends on, for instance, the mode of administration, the dosage form,and the physical properties of the active compound, such as solubilityand stability. The term “carrier” as used herein relates to atherapeutically inactive ingredient. The dosage form may be a solid,semi-solid or liquid system. The formulation may be an immediate and/ormodified release, including delayed-, sustained-, pulsed-, controlled-,targeted and programmed release formulation.

The carrier may be a liquid formulation, and is preferably a buffered,isotonic, aqueous solution. Pharmaceutically acceptable carriers alsoinclude excipients, such as diluents, carriers and the like, andadditives, such as stabilizing agents, preservatives, solubilizingagents, buffers and the like, as hereafter described.

The cyclic peptide compositions of the present invention may beformulated or compounded into pharmaceutical compositions that includeat least one cyclic peptide of the present invention together with oneor more pharmaceutically acceptable carriers, including excipients, suchas diluents, carriers and the like, and additives, such as stabilizingagents, preservatives, solubilizing agents, buffers and the like, as maybe desired. Formulation excipients may include polyvinylpyrrolidone,gelatin, hydroxy propyl cellulose (HPC), acacia, polyethylene glycol,mannitol, sodium chloride and sodium citrate. For injection or otherliquid administration formulations, water containing at least one ormore buffering constituents is preferred, and stabilizing agents,preservatives and solubilizing agents may also be employed. For solidadministration formulations, any of a variety of thickening, filler,bulking and carrier additives may be employed, such as starches, sugars,cellulose derivatives, fatty acids and the like. For topicaladministration formulations, any of a variety of creams, ointments,gels, lotions and the like may be employed. For most pharmaceuticalformulations, non-active ingredients will constitute the greater part,by weight or volume, of the preparation. For pharmaceuticalformulations, it is also contemplated that any of a variety ofmeasured-release, slow-release or sustained-release formulations andadditives may be employed, so that the dosage may be formulated so as toprovide delivery of a peptide of the present invention over a period oftime.

In general, the actual quantity of cyclic peptides of the presentinvention administered to a patient will vary between fairly wide rangesdepending on the mode of administration, the formulation used, and theresponse desired.

In practical use, the cyclic peptides of the invention can be combinedas the active ingredient in an admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, for example, oral, parenteral(including intravenous), urethral, vaginal, nasal, buccal, sublingual,or the like. In preparing the compositions for oral dosage form, any ofthe usual pharmaceutical media may be employed, such as, for example,water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents and the like in the case of oral liquid preparationssuch as, for example, suspensions, elixirs and solutions; or carrierssuch as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations such as, for example,powders, hard and soft capsules and tablets.

Because of their ease of administration, tablets and capsules representan advantageous oral dosage unit form. If desired, tablets may be coatedby standard aqueous or non-aqueous techniques. The amount of activepeptide in such therapeutically useful compositions is such that aneffective dosage will be obtained. In another dosage unit form,sublingual constructs may be employed, such as sheets, wafers, tabletsor the like.

The tablets, pills, capsules, and the like may also contain a bindersuch as povidone, gum tragacanth, acacia, corn starch or gelatin;diluents; fillers such as microcrystalline cellulose; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch or alginic acid; preservatives; colorants; a lubricant such asmagnesium stearate; and a sweetening agent such as sucrose, lactose orsaccharin. When a dosage unit form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier such as fattyoil. Various other materials may be utilized as coatings or to modifythe physical form of the dosage unit. For instance, tablets may becoated with shellac, sugar or both. A syrup or elixir may contain, inaddition to the active ingredient, sucrose as a sweetening agent, methyland propylparabens as preservatives, a dye and a flavoring such ascherry or orange flavor.

If formulated for oral delivery, the peptide is preferably formulatedand made such that it is encased in an enteric protectant, morepreferably such that it is not released until the tablet or capsule hastransited the stomach, and optionally has further transited a portion ofthe small intestine. In the context of this application it will beunderstood that the term enteric coating or material refers to a coatingor material that will pass through the stomach essentially intact butwill disintegrate after passing through the stomach to release theactive drug substance. Materials that may be used includes celluloseacetate phthalate, hydroxypropylmethyl-ethylcellulose succinate,hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, andmethacrylic acid-methyl methacrylate copolymer. The enteric coatingemployed promotes dissolution of the dosage form primarily at a siteoutside the stomach, and may be selected such that the enteric coatingdissolves at a pH of approximately at least 5.5, more preferable at a pHof from about 6.0 to about 8.0.

Any of a variety of permeation enhancers may be employed, to increaseuptake in the intestines upon dissolution of the enteric coating. In oneaspect, permeation enhancers increase either paracellular ortranscellular transport systems. Representative, non-limiting examplesof such permeation enhancers include calcium chelators, bile salts (suchas sodium cholate), and fatty acids. In some embodiments, peptides orpolypeptides that act as substrates for intestinal proteases are furtheradded. Cyclic peptides may also be administered parenterally. Solutionsor suspensions of these active peptides may for instance be prepared inwater mixed with for instance hydroxy-propylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof in oils. These preparations may optionally contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that it may be administered by syringe. The form must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, a polyol, for example glycerol,propylene glycol or liquid polyethylene glycol, suitable mixturesthereof, and vegetable oils.

The cyclic peptides of the present invention may be therapeuticallyapplied by means of nasal administration. By “nasal administration” ismeant any form of intranasal administration of any of the cyclicpeptides of the present invention. The peptides may be in an aqueoussolution, such as a solution including saline, citrate or other commonexcipients or preservatives. The peptides may also be in a dry or powderformulation.

If in an aqueous solution, the cyclic peptides may be appropriatelybuffered by means of saline, acetate, phosphate, citrate, acetate orother buffering agents, which may be at any physiologically acceptablepH, such as from about pH 4 to about pH 7. A combination of bufferingagents may also be employed, such as phosphate buffered saline, a salineand acetate buffer, and the like. In the case of saline, a 0.9% salinesolution may be employed. In the case of acetate, phosphate, citrate,and the like, a 50 mM solution may be employed. In addition to bufferingagents, a suitable preservative may be employed, to prevent or limitbacteria and other microbial growth. One such preservative that may beemployed is 0.05% benzalkonium chloride.

In an alternative embodiment, cyclic peptides of the present inventionmay be administered directly into the lung. Intrapulmonaryadministration may be performed by means of a metered dose inhaler, adevice allowing self-administration of a metered bolus of a peptide ofthe present invention when actuated by a patient during inspiration. Anyof a variety of different techniques may be used to make dry powdermicroparticles, including but not limited to micro-milling, spray dryingand a quick freeze aerosol followed by lyophilization.

The cyclic peptides of the present invention may be therapeuticallyadministered by means of an injection of a sustained releaseformulation. In general, any of a number of injectable and bioerodiblepolymers may be employed in a sustained release injectable formulation.Alternatively other sustained release formulations may be employed,including formulations permitting subcutaneous injection, which otherformulations may include one or more of nano/microspheres, liposomes,emulsions (such as water-in-oil emulsions), gels, insoluble salts orsuspensions in oil The formulation may be such that an injection isrequired on a daily, weekly, monthly or other periodic basis, dependingon the concentration and amount of cyclic peptide, the sustained releaserate of the materials employed, and other factors known to those ofskill in the art.

5.3 Routes of Administration.

If a composition including one or more peptides of the present inventionis administered by injection, the injection may be intravenous,subcutaneous, intramuscular, intraperitoneal or other means known in theart.

The peptides of the present invention may be formulated by any meansknown in the art, including but not limited to formulation as tablets,capsules, caplets, suspensions, powders, lyophilized preparations,suppositories, ocular drops, skin patches, oral soluble formulations,sprays, aerosols and the like, and may be mixed and formulated withbuffers, binders, excipients, stabilizers, anti-oxidants and otheragents known in the art. In general, any route of administration bywhich the peptides of invention are introduced across an epidermal layerof cells may be employed. Administration means may thus includeadministration through mucous membranes, buccal administration, oraladministration, dermal administration, inhalation administration, nasaladministration, urethral administration, vaginal administration, and thelike.

5.4 Therapeutically Effective Amount.

In general, the actual quantity of cyclic peptide of the presentinvention administered to a patient will vary between fairly wide rangesdepending upon the mode of administration, the patient (includingweight, sex, health condition and diet), the formulation used, and theresponse desired. The dosage for treatment is administration, by any ofthe foregoing means or any other means known in the art, of an amountsufficient to bring about the desired therapeutic effect. The cyclicpeptides of the present invention are highly active. For example, thecyclic peptide can be administered (as a single dose or in divided dailydoses) at about 0.1, 0.5, 1, 5, 50, 100, 500, 1000 or 5000 μg/kg bodyweight, depending on the specific peptide selected, the desiredtherapeutic response, the route of administration, the formulation andother factors known to those of skill in the art.

6.0 Peptides of the Present Invention

In one aspect, the present invention relates to a cyclic peptide of thestructural Formula (I):

including all enantiomers, stereoisomers or diastereoisomers thereof, ora pharmaceutically acceptable salt of any of the foregoing,

wherein:

-   -   R₁ is —NH—C(═O)— or —C(═O)—NH—;    -   R₂ is —H or —CH₂—, and if R₂ is —CH₂— forms with R₃ a        pyrrolidine ring, the pyrrolidine ring optionally substituted        with —OH;    -   R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

-   -   R_(4a), R_(4b) and R_(4c) are each independently selected from        hydrogen, halo, (C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl-dihalo,        (C₁-C₁₀)alkyl-trihalo, (C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy,        (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro, nitrile, sulfonamide,        amino, monosubstituted amino, disubstituted amino, hydroxy,        carboxy, or alkoxy-carbonyl, on the proviso that at least one of        R_(4a), R_(4b) and R_(4c) is not hydrogen;    -   R₅ is —OH or —N(R_(6a))(R_(6b));    -   R_(6a) and R_(6b) are each independently H or a C₁ to C₄ linear,        branched or cyclic alkyl chain;    -   R₇ is —H or —C(═O)—NH₂;    -   w is in each instance independently 0 to 5;    -   x is 1 to 5;    -   y is 1 to 5; and    -   z is in each instance independently 1 to 5.

In another aspect, the present invention relates to a cyclic peptide ofFormula (I) which is of formula (II):

or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention relates to a cyclic peptide ofFormula (I) which is of Formula (III):

or a pharmaceutically acceptable salt thereof.

In one aspect, the invention provides a cyclic heptapeptide whichcontains a core sequence derived from His-Phe-Arg-Trp within the cyclicportion, and where the amino acid in the first position is Arg and isoutside the cyclic portion. Without being bound by any theory, the Argresidue is believed to largely contribute to the low activity at MC1-R.

Further, the core sequence in the peptides of Formula I includes asubstituted D-Phe. Without being bound by any theory, it is believedthat the intrinsic activity may be lowered while substantiallymaintaining potency by using substituted D-Phe in peptides in accordancewith the invention. The peptide called Ref Ex 1 (including unsubstitutedD-Phe) in Table 1 can be compared with one or more of Examples 1-5,9-11, 17, 26 and 27. It may be noted that Table 1 includes mean values.

TABLE 1   0.1 ng/mL Doxycycline Intrinsic K_(i) EC₅₀ activity (nM) (nM)(%) Ref Ex 1 Ac-Arg-cyclo(Glu- 0.65 0.33 94 Dab-D-Phe-Arg- Trp-Orn)-NH₂Ref Ex 2 Ac-Arg-cyclo(Glu- 8 3.0 92 Dab-D-Phe-Arg- Trp-Orn)-OH

In a further embodiment of the invention, there are provided peptidesaccording to Formula (I), and in particular Formula (II) or (III),wherein one or more of R_(4a), R_(4b) and R_(4c) are each independentlyselected from hydrogen, halo, (C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl-dihalo,(C₁-C₁₀)alkyl-trihalo, (C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy, phenyl, nitro,nitrile, amino or hydroxy, on the proviso that at least one of R_(4a),R_(4b) and R_(4c) is not hydrogen.

In a further embodiment of the invention, there are provided peptidesaccording to Formula (I), and in particular Formula (II) or (III),wherein one or more of R_(4a), R_(4b) and R_(4c) are each independentlyselected from hydrogen, halo, (C₁-C₄)alkyl-halo, (C₁-C₄)alkyl-dihalo,(C₁-C₄)alkyl-trihalo, (C₁-C₄)alkyl, (C₁-C₄)alkoxy, phenyl, nitro,nitrile, amino or hydroxy, on the proviso that at least one of R_(4a),R_(4b) and R_(4c) is not hydrogen.

In a further embodiment of the invention, there are provided peptidesaccording to Formula (I), and in particular Formula (II) or (III),wherein one or more of R_(4a), R_(4b) and R_(4c) are each independentlyselected from

fluoro, chloro, —OH, —CH₃, —O—CH₃,

—NH₂, —NO₂ or

In a further embodiment of the invention, there are provided peptidesaccording to Formula (I), and in particular Formula (II) or (III),wherein one or more of R_(4a), R_(4b) and R_(4c) are each independentlyselected from hydrogen, halo, (C₁-C₄)alkyl-halo, (C₁-C₄)alkyl-dihalo,(C₁-C₄)alkyl-trihalo, (C₁-C₄)alkyl, (C₁-C₄)alkoxy, nitro, nitrile, aminoor hydroxy, on the proviso that at least one of R_(4a), R_(4b) andR_(4c) is not hydrogen.

In a further embodiment of the invention, there are provided peptidesaccording to Formula (I), and in particular Formula (II) or (III),wherein one or more of R_(4a), R_(4b) and R_(4c) are each independentlyselected from

fluoro, chloro, —OH, —CH₃, —O—CH₃,

—NH₂, —NO₂ or

In a further embodiment of the invention, there are provided peptidesaccording to Formula (I), and in particular Formula (II) or (III),wherein R_(4a), is selected from

fluoro, chloro, —OH, —CH₃, —O—CH₃,

—NH₂, —NO₂ or

and R_(4b) and R_(4c) are each H.

In a further embodiment of the invention, there are provided peptidesaccording to Formula (I), and in particular Formula (II) or (III),wherein R_(4a) is selected from —C≡N or —F, and R_(4b) and R_(4c) areeach H.

In a particular embodiment of the invention, there are provided peptidesaccording to Formula (I), and in particular Formula (II) or (III),wherein R_(4a) is in the 4 position and is —C≡N and R_(4b) and R_(4c)are each H.

In another particular embodiment of the invention, there are providedpeptides according to Formula (I), and in particular Formula (II) or(III), wherein R_(4a) is in the 4 position and is —F and R_(4b) andR_(4c) are each H.

The peptides encompassed within Formula (I) contain one or moreasymmetric elements such as stereogenic centers, stereogenic axes andthe like, so that the peptides encompassed within Formula (I) can existin different stereoisomeric forms. For both specific and genericallydescribed peptides, including the peptides encompassed within Formula(I), all forms of isomers at all chiral or other isomeric centers,including enantiomers and diastereomers, are intended to be coveredherein. The peptides of the invention each include multiple chiralcenters, and may be used as a racemic mixture or an enantiomericallyenriched mixture, in addition to use of the peptides of the invention inenantiopure preparations. Typically, the peptides of the invention willbe synthesized with the use of chirally pure reagents, such as specifiedL- or D-amino acids, using reagents, conditions and methods such thatenantiomeric purity is maintained, but it is possible and contemplatedthat racemic mixtures may be made. Such racemic mixtures may optionallybe separated using well-known techniques and an individual enantiomermay be used alone. In cases and under specific conditions oftemperature, solvents and pH wherein peptides may exist in tautomericforms, each tautomeric form is contemplated as being included withinthis invention whether existing in equilibrium or predominantly in oneform. Thus a single enantiomer of a peptide of Formula (I), which is anoptically active form, can be obtained by asymmetric synthesis,synthesis from optically pure precursors, or by resolution of theracemates.

The peptides of formulas (II) and (III) are specific stereoisomericforms of the peptides of Formula (I), but the invention should not beconstrued as being limited to the stereoisomeric forms encompassed byformulas (II) and (III).

The invention is further intended to include prodrugs of the presentpeptides, which on administration undergo chemical conversion bymetabolic processes before becoming active pharmacological peptides. Ingeneral, such prodrugs will be functional derivatives of the presentpeptides, which are readily convertible in vivo into a peptide ofFormula (I). Prodrugs are any covalently bonded compounds, which releasethe active parent peptide drug of Formula (I) in vivo. Conventionalprocedures for the selection and preparation of suitable prodrugderivatives are described, for example, in “Design of Prodrugs”, ed. H.Bundgaard, Elsevier, 1985. Typical examples of prodrugs havebiologically labile protecting groups on a functional moiety, such asfor example by esterification of hydroxyl, carboxyl or amino functions.Thus by way of example and not limitation, a prodrug includes peptidesof Formula (I) wherein an ester prodrug form is employed, such as, forexample, lower alkyl esters of the R group of Formula (I), such as whereR is —OH, which lower alkyl esters may include from 1-8 carbons in analkyl radical or aralkyl esters which have 6-12 carbons in an aralkylradical. Broadly speaking, prodrugs include compounds that can beoxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated,hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated,phosphorylated or dephosphorylated to produce an active parent peptidedrug of Formula (I) in vivo.

Certain modifications of peptides of Formula I may be made in order toenhance the half-life of the peptide (see G. Pasuta and F. M. Veronese(2007) “Polymer-drug conjugation, recent achievements and generalstrategies” Progress in Polymer Science 32 (8-9): 933-961).

The subject invention also includes peptides which are identical tothose recited in Formula (I), but for the fact that one or more atomsdepicted in Formula (I) are replaced by an atom having an atomic mass ormass number different from the atomic mass or mass number usually foundin nature. Examples of isotopes that can be incorporated into peptidesof the invention include isotopes of hydrogen, carbon, nitrogen andoxygen, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O and ¹⁷O, respectively.Peptides of the present invention and pharmaceutically acceptable saltsor solvates of said peptides which contain the aforementioned isotopesand/or other isotopes of other atoms are within the scope of thisinvention. Certain isotopically-labeled peptides of the presentinvention, for example those into which radioactive isotopes such as ³Hand ¹⁴C are incorporated, may have use in a variety of assays, such asin drug and/or substrate tissue distribution assays. Substitution withheavier isotopes, such as substitution of one or more hydrogen atomswith deuterium (²H), can provide pharmacological advantages in someinstances, including increased metabolic stability. Isotopically labeledpeptides of Formula (I) can generally be prepared by substituting anisotopically labeled reagent for a non-isotopically labeled reagent.

In one embodiment of the peptides according to Formula (I), and inparticular Formula (II) or (III), R₁ is —C(═O)—NH—, x is 2 and y is 3.

In another embodiment of the peptides according to Formula (I), and inparticular Formula (II) or (III), R_(i) is —C(═O)—NH—, x is 1 and y is4.

In another embodiment of the peptides according to Formula (I), and inparticular Formula (II) or (III), R₁ is —NH—C(═O)—, x is 3 and y is 2.

In still another embodiment of the peptides according to the Formula(I), and in particular Formula (II) or (III), R₁ is —NH—C(═O)—, x is 4and y is 1.

In a particular embodiment of the peptides according to the Formula (I),and in particular Formula (II) or (III), R₁ is —C(═O)—NH—, x is 2 and yis 3, or alternatively R₁ is —NH—C(═O)—, x is 3 and y is 2. Morespecifically, R₁ is —C(═O)—NH—, x is 2 and y is 3. It has surprisinglyand unexpectedly been found that peptides according to the inventionhaving a lactam bridge wherein the amide bond is positioned by means ofthe side chain of Glu (at the N-terminus end of the cyclic portion) andthe side chain of Orn (at the C-terminus end of the cyclic portion)generally provides improved potency as determined by EC₅₀, in particularin the herein described low density hMC4-R system, compared to peptideswherein the amide bond is positioned by means of the side chains of Aspand Lys, but which are otherwise identical. This discovery is contraryto assertions in the prior art that the location and direction of anamide bond in the lactam bridge of melanocortin receptor specificpeptides is of little importance for activity and does not interact withreceptors. See, for example, Bednarek M A et al., Potent and selectivepeptide agonists of α-melanotropin action at human melanocortin receptor4: their synthesis and biological evaluation in vitro. Biochem. Biophys.Res. Comm. 286:641-645 (2001).

In an embodiment of the peptides according to Formula (I), and inparticular Formula (II) or (III), R₂ is —H or —CH₂—, and if R₂ is —CH₂—forms with R₃ a pyrrolidine ring, the pyrrolidine ring optionallysubstituted with —OH;

-   -   R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

-   -   wherein w is in each instance independently selected from 0 to        5, and    -   z is in each instance independently selected from 1 to 5.

In an embodiment of the peptides according to Formula (I), and inparticular Formula (II) or (III), R₂ is —H or —CH₂—, and if R₂ is —CH₂—forms with R₃ a pyrrolidine ring, the pyrrolidine ring optionallysubstituted with —OH;

-   -   R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

-   -   wherein w is in each instance independently selected from 0 to        5, and    -   z is in each instance independently selected from 1 to 5.

In an embodiment of the peptides according to Formula (I), and inparticular Formula (II) or (III), R₂ is H.

In an embodiment of the peptides according to Formula (I), and inparticular Formula (II) or (III), R₂ is H or —CH₂—, and if R₂ is —CH₂—forms with R₃ a pyrrolidine ring, the pyrrolidine ring optionallysubstituted with —OH; and

R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

Without being bound by any theory, replacing H is by a non-aromaticamino acid, in particular a non-polar, small aliphatic amino acid, suchas Pro or Ala, or a polar uncharged or charged amino acid, such as Lys,Asp, Gln and Asn, is believed to contribute to attenuation of bloodpressure effects. Thus, the His position in the core sequence may forinstance be replaced by Dab, Dab(Acetyl), Ser, Met(O), Met(O₂), Thr,Hyp, Gln, Pro, Ala, Asn, Cit, Orn, Dap, Lys, or Arg, in particular Pro,Asn or Gln.

Thus, in a further embodiment of the invention, there are providedpeptides of Formula (I), in particular of Formula (II) or (III), whereinR₂ is —H or —CH₂—, and if R₂ is —CH₂— forms with R₃ a pyrrolidine ring,the pyrrolidine ring optionally substituted with —OH; and R₃ is —(CH₂)₂—if R₂ is —CH₂—, and otherwise R₃ is selected from

wherein w is in each instance independently selected from 0 to 5, and

z is in each instance independently selected from 1 to 5.

In a further embodiment of the invention, there are provided peptides ofFormula (I), in particular of Formula (II) or (III), wherein R₂ is —H or—CH₂—, and if R₂ is —CH₂— forms with R₃ a pyrrolidine ring, thepyrrolidine ring optionally substituted with —OH; and R₃ is —(CH₂)₂— ifR₂ is —CH₂—, and otherwise R₃ is selected from

wherein w is in each instance independently selected from 0 to 5, and

z is in each instance independently selected from 1 to 5.

In a particular embodiment of the invention, there are provided peptidesof Formula (I), in particular of Formula (II) or (III), wherein R₂ is —Hor —CH₂—, and if R₂ is —CH₂— forms with R₃ a pyrrolidine ring, thepyrrolidine ring optionally substituted with —OH; and

R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

wherein w is in each instance independently selected from 0 to 5, inparticular 0 to 2, such as 0, and

z is in each instance independently selected from 1 to 5, in particular1 to 4.

In a further particular embodiment of the invention, there are providedpeptides of Formula (I), in particular of Formula (II) or (III), whereinR₂ is —H or —CH₂—, and if R₂ is —CH₂— forms with R₃ a pyrrolidine ring,the pyrrolidine ring optionally substituted with —OH; and

R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

wherein w is in each instance independently selected from 0 to 5, inparticular 0 to 2, such as 0, and

z is in each instance independently selected from 1 to 5, in particular1 to 4.

In further embodiments of the peptides according to Formula (I), and inparticular Formula (II) or (III), R₂ is H or —CH₂—, and if R₂ is —CH₂—forms with R₃ a pyrrolidine ring, the pyrrolidine ring optionallysubstituted with —OH; and

R₃ is selected from

In another embodiment of the invention, there are provided peptides ofFormula (I), in particular of Formula (II) or (III), wherein R₂ is H andR₃ is selected from

In another embodiment of the invention, there are provided peptides ofFormula (I), in particular of Formula (II) or (III), wherein R₂ is H andR₃ is selected from

In another embodiment of the invention, there are provided peptides ofFormula (I), and in particular Formula (II) or (III), wherein R₂ is—CH₂— and R₃ is —(CH₂)₂—, R₂ and R₃ together forming an unsubstitutedpyrrolidine ring.

In still another embodiment of the invention, there are providedpeptides of Formula (I), and in particular Formula (II) or (III),wherein R₂ is —CH₂— and R₃ is —(CH₂)₂—, R₂ and R₃ together forming anpyrrolidine ring substituted with —OH. In a further embodiment of thepeptides according to Formula (I), and in particular Formula (II) or(III), R₅ is —NH₂ or —OH.

In a specific embodiment of the peptides according to Formula (I), andin particular Formula (II) or (III), R₅ is —NH₂.

One or more of the above embodiments may be combined to provide furtherspecific embodiments of the peptides according to the invention.

Thus, in a particular embodiment of the invention, there are providedpeptides according to Formula (I), in particular Formula (II) or (III),wherein

R₁ is —C(═O)—NH—, x is 2 and y is 3;

R₂ is —H or —CH₂—, and if R₂ is —CH₂— forms with R₃ a pyrrolidine ring,the pyrrolidine ring optionally substituted with —OH;

R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

w is in each instance independently selected from 0 to 5;

z is in each instance independently selected from 1 to 5;

one or more of R_(4a), R_(4b) and R_(4c) are each independently selectedfrom

fluoro, chloro, —OH, —CH₃, —O—CH₃,

—NH₂, —NO₂ or

and

R₅ is —NH₂ or —OH.

Some specific peptides of the invention are one or more of thefollowing:

(SEQ ID NO: 6) Ac-Arg-cyclo(Glu-Dab-D-Phe(2-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 7) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 8) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 9) Ac-Arg-cyclo(Glu-Dab-D-Phe(2,4-diCl)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 10) Ac-Arg-cyclo(Glu-Dab-D-Phe(3,4-diCl)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 11) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-CN)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 12) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 13) Ac-Arg-cyclo(Glu-Dab-D-Tyr-Arg-Trp-Orn)-NH₂;(SEQ ID NO: 14) Ac-Arg-cyclo(Glu-Dab-D-Phe(2-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 15) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 16) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 17) Ac-Arg-cyclo(Glu-Dab-D-Phe(2-CN)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 18) Ac-Arg-cyclo(Glu-Dab-D-Phe(2-CF₃)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 19) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-CF₃)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 20) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-Me)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 21) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-0Me)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 22) Ac-Arg-cyclo(Glu-Dab-D-Phe(3,5-diF)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 23) Ac-Arg-cyclo(Glu-Dab-D-Phe(2-Me)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 24) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-Me)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 25) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-CF₃)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 26) Ac-Arg-cyclo(Glu-Dab-D-Phe(2,4-diMe)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 27) Ac-Arg-cyclo(Glu-Dab-D-Phe(2-NO₂)-Arg-Trp-Orn- NH₂;(SEQ ID NO: 28) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-NO₂)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 29) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-NO₂)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 30) Ac-Arg-cyclo(Glu-Dab-D-Phe(3,4-diOMe)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 31) Ac-Arg-cyclo(Glu-Dab-D-Phe(3,4-diF)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 32) Ac-Arg-cyclo(Glu-Dab-D-Phe(3,4,5-triF)-Arg-Trp-Orn)-NH₂; (SEQ ID NO: 33)Ac-Arg-cyclo(Glu-Dab-D-Phe(4-NH₂)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 35)Ac-Arg-cyclo(Glu-Dab-D-Phe(4-tBu)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 36)Ac-Arg-cyclo(Glu-Ser-D-Phe(2-Cl)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 37)Ac-Arg-cyclo(Glu-Thr-D-Phe(2-Cl)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 38)Ac-Arg-cyclo(Glu-Hyp-D-Phe(2-Cl)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 39)Ac-Arg-cyclo(Glu-Gln-D-Phe(2-Cl)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 40)Ac-Arg-cyclo(Glu-Ser-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 41)Ac-Arg-cyclo(Glu-Thr-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 42)Ac-Arg-cyclo(Glu-Hyp-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 43)Ac-Arg-cyclo(Glu-Gln-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 44)Ac-Arg-cyclo(Glu-Pro-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 45)Ac-Arg-cyclo(Glu-Hyp-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 46)Ac-Arg-cyclo(Glu-Ala-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 48)Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 49)Ac-Arg-cyclo(Glu-Thr-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 50)Ac-Arg-cyclo(Glu-Ser-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 51)Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 54)Ac-Arg-cyclo(Glu-Orn-D-Phe(2-Cl)-Arg-Trp-Orn)- OH; (SEQ ID NO: 60)Ac-Arg-cyclo(Glu-Orn-D-Phe(3-Cl)-Arg-Trp-Orn)- OH; (SEQ ID NO: 61)Ac-Arg-cyclo(Glu-Dab(Acetyl)-D-Phe(2-Cl)-Arg- Trp-Orn)-NH₂;(SEQ ID NO: 63) Ac-Arg-cyclo(Glu-Ser-D-Phe(2-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 64) Ac-Arg-cyclo(Glu-Gln-D-Phe(2-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 65) Ac-Arg-cyclo(Glu-Hyp-D-Phe(4-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 66) Ac-Arg-cyclo(Glu-Dap-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 67) Ac-Arg-cyclo(Glu-Dap-D-Phe(2-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 69) Ac-Arg-cyclo(Glu-Hyp-D-Phe(2-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 70) Ac-Arg-cyclo(Glu-Ser-D-Phe(4-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 71) Ac-Arg-cyclo(Glu-Pro-D-Phe(2-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 72) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 73) Ac-Arg-cyclo(Glu-Met(O₂)-D-Phe(4-F)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 74) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 78) Ac-Arg-cyclo(Glu-Asn-D-Phe(2-Me)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 81) Ac-Arg-cyclo(Glu-Asn-D-Phe(3,5-diF)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 89) Ac-Arg-cyclo(Glu-Asn-D-Phe(2-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 90) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)- OH;(SEQ ID NO: 91) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)- OH;(SEQ ID NO: 92) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-OMe)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 93) Ac-Arg-cyclo(Glu-Pro-D-Phe(2-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 97) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 100) Ac-Arg-cyclo(Glu-Asn-D-Phe(3,4,5-triF)-Arg-Trp-Orn)-NH₂; (SEQ ID NO: 102)Ac-Arg-cyclo(Glu-Gln-D-Phe(3,4,5-triF)-Arg-Trp- Orn)-NH₂;(SEQ ID NO: 104) Ac-Arg-cyclo(Glu-Arg-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 105) Ac-Arg-cyclo(Glu-Lys-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 106) Ac-Arg-cyclo(Glu-Orn-D-Phe(3-Cl)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 107) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)- OH;(SEQ ID NO: 111) Ac-Arg-cyclo(Glu-Asp-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 112) Ac-Arg-cyclo(Glu-Asp-D-Phe(4-CN)-Arg-Trp-Orn)- OH;(SEQ ID NO: 113) Ac-Arg-cyclo(Glu-Glu-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 114) Ac-Arg-cyclo(Glu-Glu-D-Phe(4-CN)-Arg-Trp-Orn)- OH;(SEQ ID NO: 115) Ac-Arg-cyclo(Glu-Gln-D-Phe(2-Me)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 119) Ac-Arg-cyclo(Glu-Gln-D-Phe(2-CF₃)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 121) Ac-Arg-cyclo(Glu-Asn-D-Phe(2-CF₃)-Arg-Trp-Orn)- NH₂;or a pharmaceutically acceptable salt of any of the foregoing.

Particularly, the invention relates to:

(SEQ ID NO: 48) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 74) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-F)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 90) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)- OH,or a pharmaceutically acceptable salt of any of the foregoing.

Particularly, the invention also relates to:

(SEQ ID NO: 51) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 91) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)- OH;(SEQ ID NO: 97) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-F)-Arg-Trp-Orn)- NH₂,or a pharmaceutically acceptable salt of any of the foregoing.

Particularly, the invention also relates to:

(SEQ ID NO: 44) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂;(SEQ ID NO: 72) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)- NH₂,(SEQ ID NO: 107) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)- OH;or a pharmaceutically acceptable salt of any of the foregoing.

Some further specific peptides of the invention are:

(SEQ ID NO: 75) Ac-Arg-cyclo(Orn-Asn-D-Phe(4-F)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 76) Ac-Arg-cyclo(Orn-Asn-D-Phe(2-F)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 77) Ac-Arg-cyclo(Orn-Asn-D-Phe(3-Me)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 79) Ac-Arg-cyclo(Orn-Asn-D-Phe(2-Me)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 80) Ac-Arg-cyclo(Orn-Asn-D-Phe(3,4-diF)-Arg-Trp- Glu)-NH₂;(SEQ ID NO: 82) Ac-Arg-cyclo(Orn-Asn-D-Phe(3,5-diF)-Arg-Trp- Glu)-NH₂;(SEQ ID NO: 96) Ac-Arg-cyclo(Orn-Asn-D-Phe(4-CN)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 99) Ac-Arg-cyclo(Orn-Gln-D-Phe(4-CN)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 101) Ac-Arg-cyclo(Orn-Asn-D-Phe(3,4,5-triF)-Arg-Trp-Glu)-NH₂; (SEQ ID NO: 103)Ac-Arg-cyclo(Orn-Gln-D-Phe(3,4,5-triF)-Arg-Trp- Glu)-NH₂;(SEQ ID NO: 110) Ac-Arg-cyclo(Orn-Pro-D-Phe(2-F)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 116) Ac-Arg-cyclo(Orn-Gln-D-Phe(2-Me)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 117) Ac-Arg-cyclo(Orn-Gln-D-Phe(2-Cl)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 118) Ac-Arg-cyclo(Orn-Asn-D-Phe(2-Cl)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 120) Ac-Arg-cyclo(Orn-Gln-D-Phe(2-CF₃)-Arg-Trp-Glu)- NH₂;(SEQ ID NO: 122) Ac-Arg-cyclo(Orn-Asn-D-Phe(2-CF₃)-Arg-Trp-Glu)- NH₂;or a pharmaceutically acceptable salt of any of the foregoing.

6.1 Specific Peptides.

Peptides of the following structures were synthesized by the generalmethods described above, and except where indicated MC1-R Ki and MC4-RKi values for each peptide were determined in competitive binding assaysusing [I¹²⁵]-NDP-α-MSH as described in 7.1 below. All peptides wereprepared in the TFA acid salt form, except for the peptides of Examples43, 46 and 67, which were prepared in the acetate salt form.

The syntheses of some specific peptides of the invention are illustratedbelow. These peptides were prepared using solid phase peptide synthesisby means of a Symphony Multiplex Peptide Synthesizer (Rainin InstrumentCompany/Protein Technologies Inc) automated peptide synthesizer.

Step 1: Coupling of Orn

The Sierber resin 9-Fmoc-Aminoxanthen-3-yloxy-polystyrene resin (0.39mol/g, ChemPep Inc., #151902) was swelled in 3×5 mL of DMF for 10 min.Thereafter, Fmoc was deprotected using 2×5 mL of 20% piperidine in DMFfor 10 min. The resin was then washed in 6×5 mL DMF for 30 sec. 5 mL of200 mM Fmoc-Orn(Alloc)-OH in DMF and 5 mL 200 mM HBTU containing 400 mMNMM in DMF was added and after 30 min the resin was washed with 3×5 mLDMF for 30 sec.

Step 2: Coupling of Next 6 Amino Acids (AA)

The resin from step 1 was first swelled in 3×5 mL of DMF for 30 sec,Fmoc was deprotected using 2×5 mL of 20% piperidine in DMF for 10 minand then washed with 6×5 mL DMF for 30 sec. 5 mL of 200 mM Fmoc-AA-OHsolution and 5 mL 200 mM HBTU containing 400 mM NMM in DMF was added andafter 30 min the resin was washed with 3×5 mL DMF for 30 sec.

This step was repeated for each amino acid (AA).

Step 3: Acetylation

Fmoc was deprotected using 2×5 mL of 20% piperidine in DMF for 10 minand the resin was then washed with 3×5 mL DMF for 30 sec. Thereafter, 5mL of 50% Ac₂O/DMF solution was added and after 30 min the peptide resinwas washed with 3×5 mL DMF for 30 sec and 6×5 mL DCM for 30 sec.

Step 4: Allyl/Alloc Deprotection

The peptide resin (0.6 mmol) was mixed with phenylsilane (OakwoodChemical, #S13600) (20 eq.) in 20 mL of DCM and bubbled with nitrogenfor 5 min.

Tetrakis(triphenylphosphine)-palladium(0) (Strem Chemicals, Inc.,#46-2150) (0.2 eq.) was added and the mixture was agitated with nitrogenfor 1 hour. The procedure was repeated one time for 1 hour and anadditional time for 30 min with fresh reagents. The treated peptideresin was then washed with DCM×3 and DMF×3.

Step 5: Lactam Formation

The lactam ring was formed on the peptide resin using TBTU (2 eq.) andethyldiisopropylamine (DIEA) (4 eq.) in 20 mL DMF for 1 hour. A secondcoupling may be needed if a positive Kaiser Ninhydrin test is observed.

Step 6: Peptide Cleavage

The peptide resin (0.6 mmol) was mixed with 20 mL of 5% sodiumdiethyldithio-carbamate trihydrate (NaCS₂NEt₂, Aldrich, #228680) in DMFfor 20 min and then washed with DMF×3, DCM×3 and diethyl ether×2.

The resin (0.6 mmol) was then stirred in a 25 mL of TFA/TIS/H2O(90:5.0:5.0 v/v/v) for 2.5 hours. The resin was filtered. The filtratewas concentrated to about 10 mL in volume and about 140 mL of colddiethyl ether (pre-cooled to about 0° C.) was added.

The mixture was vortexed, and then placed in the refrigerator (about −4°C.) for 1 h, centrifuged for 5 min at 2800 rpm, and the ether layer wasdecanted.

The peptide was washed with 90 mL of cold diethyl ether (pre-cooled toabout 0° C.), vortexed, centrifuged for 5 min at 2800 rpm, and etherlayer decanted.

The resulting solid was dissolved in 50% AcOH/H₂O and stored at roomtemperature overnight.

The crude peptide solution was concentrated to afford solid crudepeptide for HPLC purification.

After HPLC purification, the peptide TFA salt was converted to peptideacetate salt using ion exchange (×100 eq.). The anion exchange resinused was Dowex SBR LC NG, OH-form (Supelco, Cat# 14036-U).

Example 46 Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)-NH₂ (SEQ IDNO:51)

The procedure described above was followed in the preparation of thetitle peptide.

The amino acids added in step 2 were, in the order of being coupled,Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe(4-CN)—OH,Fmoc-Gln(Trt)-OH, Fmoc-Glu(OAll)-OH, and Fmoc-Arg(Pbf)-OH.

The resulting peptide was purified by HPLC (column: Atlantis dC18 OBD™19×100 mm (5μ, Waters part #186001367) using 10% MeOH/H₂O containing0.1% TFA (solvent A) and 90% MeOH/H₂O containing 0.1% TFA (solvent B). Agradient of 0%-5% of solvent B for 5 min and 5%-35% of solvent B for 30min was used.

The peptide yield was 10%.

Example 67 Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-NH₂ (SEQ IDNO:72)

The procedure described above was followed in the preparation of thetitle peptide except for that 15 mL of 5% sodium diethyldithio-carbamatetrihydrate in DMF, 16 mL of TFA/TIS/H₂O and 90 mL+60 mL of diethyl etherwere used in step 6. Moreover, the filtrate was concentrated to 5 mL.

The amino acids added in step 2 were, in the order of being coupled,Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe(4-F)—OH, Fmoc-Pro-OH,Fmoc-Glu(OAll)-OH, and Fmoc-Arg(Pbf)-OH.

The resulting peptide was purified by HPLC (column: Atlantis dC18 OBD™19×100 mm (5μ, Waters part #186001367) using 10% MeOH/H₂O containing0.1% TFA (solvent A) and 90% MeOH/H₂O containing 0.1% TFA (solvent B). Agradient of 5%-10% of solvent B for 5 min and 10%-40% of solvent B for30 min was used.

The peptide yield was 16%.

MC4- MC1- R Ki R Ki No. Structure Amino Acid Sequence (nM) (nM) 1

Ac-Arg-cyclo(Glu-Dab- D-Phe(2-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 6) 3 1 2

Ac-Arg-cyclo(Glu-Dab- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 7) 0.751 3

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 8) 0.080.3 4

Ac-Arg-cyclo(Glu-Dab- D-Phe(2,4-diCl)-Arg- Trp-Orn)-NH₂ (SEQ ID NO: 9)0.055 0.5 5

Ac-Arg-cyclo(Glu-Dab- D-Phe(3,4-diCl)-Arg- Trp-Orn)-NH₂ (SEQ ID NO: 10)0.055 0.2 6

Ac-Arg-cyclo(Glu-Dab- D-Phe(3-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 11) 3343 7

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 12)0.833 5 8

Ac-Arg-cyclo(Glu-Dab- D-Tyr-Arg-Trp-Orn- NH₂ (SEQ ID NO: 13) 75 15 9

Ac-Arg-cyclo(Glu-Dab- D-Phe(2-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 14) 0.70.8 10

Ac-Arg-cyclo(Glu-Dab- D-Phe(3-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 15 2 2 11

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 16) 0.250.5 12

Ac-Arg-cyclo(Glu-Dab- D-Phe(2-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 17) 4535 13

Ac-Arg-cyclo(Glu-Dab- D-Phe(2-CF₃)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 18) 0.63 14

Ac-Arg-cyclo(Glu-Dab- D-Phe(3-CF₃)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 19)0.95 9 15

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-Me)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 20)0.145 0.75 16

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-OMe)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 21)0.25 0.15 17

Ac-Arg-cyclo(Glu-Dab- D-Phe(3,5-diF)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 22) 2— 18

Ac-Arg-cyclo(Glu-Dab- D-Phe(2-Me)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 23) 0.5— 19

Ac-Arg-cyclo(Glu-Dab- D-Phe(3-Me)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 24) 0.4— 20

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-CF₃)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 25)0.06 — 21

Ac-Arg-cyclo(Glu-Dab- D-Phe(2,4-diMe)-Arg- Trp-Orn)-NH₂ (SEQ ID NO: 26)0.085 — 22

Ac-Arg-cyclo(Glu-Dab- D-Phe(2-NO₂)-Arg-Trp- Orn-NH₂ (SEQ ID NO: 27) 11 —23

Ac-Arg-cyclo(Glu-Dab- D-Phe(3-NO₂)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 28) 0.7— 24

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-NO₂)-Arg-Trfp- Orn)-NH₂ (SEQ ID NO: 29)0.45 — 25

Ac-Arg-cyclo(Glu-Dab- D-Phe(3,4-diOMe)-Arg- Trp-Orn)-NH₂ (SEQ ID NO: 30)35 — 26

Ac-Arg-cyclo(Glu-Dab- D-Phe(3,4-diF)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 31)0.3 — 27

Ac-Arg-cyclo(Glu-Dab- D-Phe(3,4,5-triF)-Arg- Trp-Orn)-NH₂ (SEQ ID NO:32) 0.667 — 28

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-NH₂)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 33) 48— 29

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-Ph)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 34) 0.5— 30

Ac-Arg-cyclo(Glu-Dab- D-Phe(4-tBu)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 35) 4 —31

Ac-Arg-cyclo(Glu-Ser- D-Phe(2-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 36) 8300 32

Ac-Arg-cyclo(Glu-Thr- D-Phe(2-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 37) 11540 33

Ac-Arg-cyclo(Glu-Hyp- D-Phe(2-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 38) 1143 34

Ac-Arg-cyclo(Glu-Gln- D-Phe(2-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 39) 7128 35

Ac-Arg-cyclo(Glu-Ser- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 40) 10810 36

Ac-Arg-cyclo(Glu-Thr- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 41) 6380 37

Ac-Arg-cyclo(Glu-Hyp- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 42) 2430 38

Ac-Arg-cyclo(Glu-Gln- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 43) 3290 39

Ac-Arg-cyclo(Glu-Pro- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 44) 41550 40

Ac-Arg-cyclo(Glu-Hyp- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 45) 2880 41

Ac-Arg-cyclo(Glu-Ala- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 46) 34300 42

Ac-Arg-cyclo(Glu-Val- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 47) 6420 43

Ac-Arg-cyclo(Glu-Asn- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 48) 5870 44

Ac-Arg-cyclo(Glu-Thr- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 49) 181625 45

Ac-Arg-cyclo(Glu-Ser- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 50) 1010000 46

Ac-Arg-cyclo(Glu-Gln- D-Phe(4-CN)-Arg-Thr- Orn)-NH₂ (SEQ ID NO: 51) 4425 47

Ac-Arg-cyclo(Glu- Ser(Bzl)-D-Phe(2-Cl)- Arg-Trp-Orn)-NH₂ (SEQ ID NO: 52)0.3 25 48

Ac-Arg-cyclo(Glu- Phe(3-C(═O)—NH₂)-D- Phe(2-Cl)-D-Phe-Arg- Trp-Orn)-NH₂(SEQ ID NO: 53) 15 545 49

Ac-Arg-cyclo(Glu-Orn- D-Phe(2-Cl)-Arg-Trp- Orn)-OH (SEQ ID NO: 54) 8 850

Ac-D-Arg-cyclo(Orn- His-D-Phe(2-Cl)-Arg- Trp-Glu)-NH₂ (SEQ ID NO: 55) 12 51

Ac-D-Arg-cyclo(Orn- His-D-Phe(3-Cl)-Arg- Trp-Glu)-NH₂ (SEQ ID NO: 56)0.6 3 52

Ac-D-Arg-cyclo(Orn- His-D-Phe(4-CN)-Arg- Trp-Glu)-NH₂ (SEQ ID NO: 57)0.6 6 53

Ac-D-Arg-cyclo(Orn- His-D-Phe(2-F)-Arg- Trp-Glu)-NH₂ (SEQ ID NO: 58)0.95 0.75 54

Ac-D-Arg-cyclo(Orn- His-D-Phe(3,4-diF)-Arg- Trp-Glu)-NH₂ (SEQ ID NO: 59)0.25 0.7 55

Ac-Arg-cyclo(Glu-Orn- D-Phe(3-Cl)-Arg-Trp- Orn)-OH (SEQ ID NO: 60) 20 2356

Ac-Arg-cyclo(Glu- Dab(Acetyl)-D-Phe(2- Cl)-Arg-Trp-Orn)-NH₂ (SEQ ID NO:61) 11 30 57

Ac-D-Arg-cyclo(Orn- His-D-Phe(4-F)-Arg- Trp-Glu)-NH₂ (SEQ ID NO: 62)0.65 1 58

Ac-Arg-cyclo(Glu-Ser- D-Phe(2-F-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 63) 15 13859

Ac-Arg-cyclo(Glu-Gln- D-Phe(2-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 64) 7 5860

Ac-Arg-cyclo(Glu-Hyp- D-Phe(4-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 65) 1 8961

Ac-Arg-cyclo(Glu-Dap- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 66) 4 2562

Ac-Arg-cyclo(Glu-Dap- D-Phe(2-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 67) 9 2863

Ac-Arg-cyclo(Glu- Thr(Bzl)-D-Phe(2-Cl)- Arg-Trp-Orn)-NH₂ (SEQ ID NO: 68)0.35 12 64

Ac-Arg-cyclo(Glu-Hyp- D-Phe(2-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 69) 3 5365

Ac-Arg-cyclo(Glu-Ser- D-Phe(4-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 70) 5 4366

Ac-Arg-cyclo(Glu-Pro- D-Phe(2-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 71) 6 9567

Ac-Arg-cyclo(Glu-Pro- D-Phe(4-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 72) 5 13068

Ac-Arg-cyclo(Glu- Met(O₂)-D-Phe(4-F)- Arg-Trp-Orn)-NH₂ (SEQ ID NO: 73) 233 69

Ac-Arg-cyclo(Glu-Asn- D-Phe(4-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 74) 3 15070

Ac-Arg-cyclo(Orn-Asn- D-Phe(4-F)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 75) 3 17071

Ac-Arg-cyclo(Orn-Asn- D-Phe(2-F)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 76) 9 33572

Ac-Arg-cyclo(Orn-Asn- D-Phe(3-Me)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 77) 42300 73

Ac-Arg-cyclo(Glu-Asn- D-Phe(2-Me)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 78) 4160 74

Ac-Arg-cyclo(Orn-Asn- D-Phe(2-Me)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 79) 5405 75

Ac-Arg-cyclo(Orn-Asn- D-Phe(3,4-diF)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 80) 3190 76

Ac-Arg-cyclo(Glu-Asn- D-Phe(3,5-diF)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 81)20 260 77

Ac-Arg-cyclo(Orn-Asn- D-Phe(3,5-diF)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 82) 8190 78

Ac-D-Arg-cyclo(Asp- Gln-D-Phe(3,4-diF)-Arg- Trp-Lys)-NH₂ (SEQ ID NO: 83)8 515 79

Ac-D-Arg-cyclo(Asp- Gln-D-Phe(2-Cl)-Arg- Trp-Lys)-NH₂ (SEQ ID NO: 84) 10395 80

Ac-D-Arg-cyclo(Asp- Gln-D-Phe(3-Cl)-Arg- Trp-Lys)-NH₂ (SEQ ID NO: 85) 71750 81

Ac-D-Arg-cyclo(Asp- Gln-D-Phe(4-CN)-Arg- Trp-Lys)-NH₂ (SEQ ID NO: 86) 1310000 82

Ac-D-Arg-cyclo(Asp- Gln-D-Phe(2-F)-Arg- Trp-Lys)-NH₂ (SEQ ID NO: 87) 10220 83

Ac-D-Arg-cyclo(Asp- Gln-D-Phe(4-F)-Arg- Trp-Lys)-NH₂ (SEQ ID NO: 88) 6260 84

Ac-Arg-cyclo(Glu-Asn- D-Phe(2-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 89) 7245 85

Ac-Arg-cyclo(Glu-Asn- D-Phe(4-CN)-Arg-Trp- Orn)-OH (SEQ ID NO: 90) 6410000 86

Ac-Arg-cyclo(Glu-Gln- D-Phe(4-CN)-Arg-Trp- Orn)-OH (SEQ ID NO: 91) 403968 87

Ac-Arg-cyclo(Glu-Dab- D-Phe(3-OMe)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 92) 3 —88

Ac-Arg-cyclo(Glu-Pro- D-Phe(2-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 93) 5 —89

Ac-Arg-cyclo(Asp-Asn- D-Phe(4-CN)-Arg-Trp- Lys)-NH₂ (SEQ ID NO: 94) 48 —90

Ac-Arg-cyclo(Asp-Gln- D-Phe(4-CN)-Arg-Trp- Lys)-NH₂ (SEQ ID NO: 95) 5 —91

Ac-Arg-cyclo(Orn-Asn- D-Phe(4-CN)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 96) 4 —92

Ac-Arg-cyclo(Glu-Gln- D-Phe(4-F)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 97) 2 —93

Ac-Arg-cyclo(Lys-Gln- D-Phe(4-CN)-Arg-Trp- Asp)-NH₂ (SEQ ID NO: 98) 1 —94

Ac-Arg-cyclo(Orn-Gln- D-Phe(4-CN)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 99) 0.9— 95

Ac-Arg-cyclo(Glu-Asn- D-Phe(3,4,5-triF)-Arg- Trp-Orn)-NH₂ (SEQ ID NO:100) 3 — 96

Ac-Arg-cyclo(Orn-Asn- D-Phe(3,4,5-triF)-Arg- Trp-Glu)-NH₂ (SEQ ID NO:101) 2 — 97

Ac-Arg-cyclo(Glu-Gln- D-Phe(3,4,5-triF)-Arg- Trp-Orn)-NH₂ (SEQ ID NO:102) 0.75 — 98

Ac-Arg-cyclo(Orn-Gln- D-Phe(3,4,5-triF)-Arg- Trp-Glu)-NH₂ (SEQ ID NO:103) 0.75 — 99

Ac-Arg-cyclo(Glu-Arg- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 104) 0.5— 100

Ac-Arg-cyclo(Glu-Lys- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 105) 4 —101

Ac-Arg-cyclo(Glu-Orn- D-Phe(3-Cl)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 106) 1 —102

Ac-Arg-cyclo(Glu-Pro- D-Phe(4-F)-Arg-Trp- Orn)-OH (SEQ ID NO: 107) 30730 103

Ac-Arg-cyclo(Asp-Pro- D-Phe(4-F)-Arg-Trp- Lys)-NH₂ (SEQ ID NO: 108) 4 —104

Ac-Arg-cyclo(Asp-Pro- D-Phe(2-F)-Arg-Trp- Lys)-NH₂ (SEQ ID NO: 109) 78 —105

Ac-Arg-cyclo(Orn-Pro- D-Phe(2-F)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 110) 7 —106

Ac-Arg-cyclo(Glu-Asp- F-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 111) 64710000 107

Ac-Arg-cyclo(Glu-Asp- D-Phe(4-CN)-Arg-Trp- Orn)-OH (SEQ ID NO: 112) 646310000 108

Ac-Arg-cyclo(Glu-Glu- D-Phe(4-CN)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 113) 12810000 109

Ac-Arg-cyclo(Glu-Glu- D-Phe(4-CN)-Arg-Trp- Orn)-OH (SEQ ID NO: 114) 109010000 110

Ac-Arg-cyclo(Glu-Gln- D-Phe(2-Me)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 115) 5105 111

Ac-Arg-cyclo(Orn-Gln- D-Phe(2-Me)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 116) 6145 112

Ac-Arg-cyclo(Orn-Gln- D-Phe(2-Cl)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 117) 483 113

Ac-Arg-cyclo(Orn-Asn- D-Phe(2-Cl)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 118) 7255 114

Ac-Arg-cyclo(Glu-Gln- D-Phe(2-CF₃)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 119) 893 115

Ac-Arg-cyclo(Orn-Gln- D-Phe(2-CF₃)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 120) 4235 116

Ac-Arg-cyclo(Glu-Asn- D-Phe(2-CF₃)-Arg-Trp- Orn)-NH₂ (SEQ ID NO: 121) 7380 117

Ac-Arg-cyclo(Orn-Asn- D-Phe(2-CF₃)-Arg-Trp- Glu)-NH₂ (SEQ ID NO: 122) 5540

7.0 Tests and Assays Employed in Evaluation of the Peptides of thePresent Invention

The melanocortin receptor-specific peptides of this invention may betested by a variety of assay systems and animal models to determinebinding, functional status and efficacy.

7.1 Competitive Inhibition Assay Using [I¹²⁵]-NDP-α-MSH.

A competitive inhibition binding assay was performed for exemplifiedpeptides according to the invention using membrane homogenates preparedfrom HEK-293 cells that express recombinant hMC4-R, hMC3-R, or hMC5-R,and from B-16 mouse melanoma cells (containing endogenous MC1-R). Insome instances, HEK-293 cells that express recombinant hMC1-R wereemployed. Assays were performed in 96 well GF/B Millipore multiscreenfiltration plates (MAFB NOB10) pre-coated with 0.5% bovine serum albumin(Fraction V). Membrane homogenates were incubated with 0.2 nM (forhMC4-R) 0.4 nM (for MC3-R and MC5-R) or 0.1 nM (for mouse B16 MC1-R orhMC1-R) [I¹²⁵]-NDP-α-MSH (Perkin Elmer) and increasing concentrations oftest peptides of the present invention in buffer containing 25 mM HEPESbuffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.3 mM1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for60 minutes at 37° C., the assay mixture was filtered and the membraneswashed three times with ice-cold buffer. Filters were dried and countedin a gamma counter for bound radioactivity. Non-specific binding wasmeasured by inhibition of binding of [I¹²⁵]-NDP-α-MSH in the presence of1 μM NDP-α-MSH. Maximal specific binding (100%) was defined as thedifference in radioactivity (cpm) bound to cell membranes in the absenceand presence of 1 μM NDP-α-MSH. Radioactivity (cpm) obtained in thepresence of test peptides was normalized with respect to 100% specificbinding to determine the percent inhibition of [I¹²⁵]-NDP-α-MSH binding.Each assay was conducted in triplicate and the actual mean values aredescribed, with results less than 0% reported as 0%. Ki values for testpeptides of the present invention were determined using Graph-Pad Prism®curve-fitting software. Results from this assay are presented herein(see 6.1). For some peptides, a mean value of more than two values ispresented.

7.2 Assay for Agonist Activity.

Accumulation of intracellular cAMP was examined as a measure of theability of peptides of the present invention to elicit a functionalresponse in HEK-293 cells that is express MC4-R. Confluent HEK-293 cellsthat express recombinant hMC4-R were detached from culture plates byincubation in enzyme-free cell dissociation buffer. Dispersed cells weresuspended in Earle's Balanced Salt Solution containing 10 mM HEPES (pH7.5), 1 mM MgCl₂, 1 mM glutamine, 0.5% albumin and 0.3 mM3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. Thecells were plated in 96-well plates at a density of 0.5×10⁵ cells perwell and pre-incubated for 10 minutes. Cells were exposed for 15 minutesat 37° C. to peptides of the present invention dissolved in DMSO (finalDMSO concentration of 1%) at a concentration range of 0.05-5000 nM in atotal assay volume of 200 pt. NDP-α-MSH was used as the referenceagonist. cAMP levels were determined by an HTRF® cAMP cell-based assaysystem from Cisbio Bioassays utilizing cryptate-labeled anti-cAMP andd2-labeled cAMP, with plates read on a Perkin-Elmer Victor plate readerat 665 and 620 nM. Data analysis was performed by nonlinear regressionanalysis with Graph-Pad Prism® software. The maximum efficacies of testpeptides of the present invention were compared to that achieved by thereference melanocortin agonist NDP-α-MSH, used as full agonist benchmarkin this context.

7.3 High and Low Density hMC4-R Functional Assay.

A HEK293 cell line transfected with human MC4-R (Palatin Technologies,US, with license from the University of Michigan) was used. The humanMC4-R was introduced to HEK293 by using the T-REx™ System, Invitrogen.The T-REx™ System employs a tetracycline-regulated mammalian expressionsystem that uses regulatory elements from the E. coli Tn10-encodedtetracycline (Tet) resistance operon. By use of the T-REx™ System,expression of the gene of interest, the human MC4-R gene, is repressedin the absence of tetracycline or doxycycline and induced in thepresence of tetracycline or doxycycline (see T-REx™ System Manual,published by Invitrogen).

HEK293-T-REx-MC4-R cells were cultured in DMEM (Gibco 11965),supplemented with L-Glutamine (Gibco 25030), 10% fetal bovine serum(FBS), 200 μg/mL Zeocin (Invitrogen 46-0072) and 6 μg/mL Blasticidin(Invitrogen 46-1120) in 5% CO₂ and 95% humidity at 37° C. T-150 flasksof cells at 75% confluence were incubated with two concentrations ofdoxycycline (0.1 ng/mL to provide a low density hMC4-R system and 10ng/mL to provide a high density hMC4-R system) in 5% CO₂ at 37° C. for16-18 hours to induce MC4-R expression. On the day of the assay, thecells were washed with PBS (Gibco 14190) and harvested using celldissociation buffer (Gibco 13150-016), then centrifuged and resuspendedin Hanks' Balanced Salt Solution (+Ca, +Mg) (Gibco 14025), 10 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4)(Sigma H0887), 1 mM L-Glutamine (Gibco 25030), 1 mg/mL bovine serumalbumin (BSA) (Sigma A3311) and 0.3 mM 3-isobutyl-1-methyl-xanthine(IBMX). Cells were then counted and volume was adjusted to 2.5×10⁵ cellsper ml.

The cells were then dispensed into 96-well plates (BD 353916) in 198 μL(about 5×10⁴) cells/well and incubated for 10 minutes at 37° C. Thecompound to be tested was diluted with DMSO to a final concentration of1 mM. Serial dilution was prepared in polypropylene removable 12-welllibrary tube strips (VWR cat#83009-682). 120 μL of the 1 mM compoundstock was pipetted in the second column on the plate. Using the Janusliquid handler the compound was serially diluted 1:10 (25 μLcompound+225 μL DMSO) to a total of 9 concentrations (representing finalassay concentrations ranging from 10⁻⁵ to 10⁻¹³ M).

2 μL of the standard, [Nle⁴, D-Phe⁷]-alpha-Melanocyte StimulatingHormone (NDP-α-MSH), or compound was added to the 96-well plate usingthe Janus robotic system. All assay samples were run in duplicate (i.e.each sample was in two low dox and two high dox plates, respectively).The plates were gently shaken and incubated for 15 minutes at 37° C. Thereaction was stopped by adding 15 μL of lysis buffer per well and theplates were shakened for 30 minutes at room temperature.

Agonist stimulation of the MC4-R activates adenylate cyclase, which isan enzyme that catalyses the formation 3′,5′-cyclic adenosinemonophosphate (cAMP) from adenosine triphosphate (ATP). Thus, agoniststimulation of the MC4-R increases the levels of cAMP. cAMP-levels weremeasured with the cAMP dynamic 2 HTRF kit (CisBio cat#62AM4PEC; seemanual published by CisBio). cAMP levels were normalised against platecontrols (1% DMSO for 0%, 400 nM NDP-α-MSH for 100%) and a calibrationcurve ranging from 712 nM to 0.04 nM cAMP (essentially as described inthe CisBio HTRF kit). The plates were incubated on a shaker at roomtemperature for 1 hour and read on the Perkin-Elmer Victor plate readerat 665 and 620 nm. Fluoresence ratios were then calculated as describedin the CisBio HTRF kit, with GraphPad Prism software used to plot thechange in fluorescence percent values versus cAMP concentration usingthe variable slope dose response curve and, based on calculated cAMPconcentrations, to determine EC₅₀ values and percent activation. Resultsfrom this assay are presented herein (see 8.0).

7.4 Food Intake and Body Weight Change.

Change in food intake and body weight was evaluated for selectedpeptides administered by subcutaneous injection route. MaleSprague-Dawley rats were obtained from Hilltop Lab Animals, Inc.(Scottdale, Pa.). Animals were individually housed in conventionalpolystyrene hanging cages and maintained on a controlled 12 hour on/offlight cycle. Water and pelleted food (ProLab RMH 2500, W.F. Fisher & SonInc.) was provided ad libitum. The rats were dosed subcutaneously withvehicle (3.2% mannitol/50 mM Tris buffer) or selected peptides (1.0mg/kg). The changes in food intake for the 4 and 24 hour periods afterdosing and the changes in body weight for the 24 hour period afterdosing were determined. The changes in body weight and food intake forthe 48 hour and 72 hour periods after dosing was also measured todetermine reversal of changes in body weight and food intake effectsback to baseline levels (not shown).

Food intake (FI, 0-4 h and 0-24 h) and body weight (BW, 0-24 h) resultsfrom studies with 1.0 mg/kg subcutaneously of Examples 43, 46 and 67 andcorresponding vehicle controls run in parallel are shown in Table 2below, expressed as means±SEM (n=8-10).

TABLE 2 Dose BW change Experiment Treatment (mg/kg, s.c.) n FI 0-4 h (g)FI 0-24 h (g) 0-24 h (g) 1 Vehicle 10 10.53 ± 0.78  27.75 ± 1.00 1.49 ±0.18 1 Ref Ex 2 1.0 10 4.41 ± 0.48 20.84 ± 1.01 −0.38 ± 0.46  1 Ex 431.0 10 6.85 ± 0.55 24.44 ± 0.85 0.45 ± 0.35 2 Vehicle 8 7.09 ± 0.3927.46 ± 1.16 1.96 ± 0.55 2 Ref Ex 2 1.0 8 2.80 ± 0.27 21.81 ± 1.04 0.85± 0.45 2 Ex 46 1.0 8 3.71 ± 0.68 20.56 ± 1.14 −0.29 ± 0.55  3 Vehicle 87.76 ± 0.53 25.71 ± 0.25 2.25 ± 0.50 3 Ref Ex 2 1.0 8 3.17 ± 0.42 19.98± 0.60 −0.27 ± 0.48  3 Ex 67 1.0 8 4.20 ± 0.66 21.51 ± 1.27 −0.05 ±0.39 

7.5 Induction of Penile Erection.

The ability of peptides of the present invention to induce penileerection (PE) in male rats was evaluated with selected peptides. MaleSprague-Dawley rats weighing 250-300 g were kept on a 12 hour on/offlight cycle with food and water ad libitum. All behavioral studies wereperformed between 9 a.m. and 4 p.m. Groups of 6-8 rats were administeredpeptides at a variety of doses via the subcutaneous injection route.Immediately after treatment, rats were placed into individualpolystyrene cages (27 cm long, 16 cm wide, and 25 cm high) forbehavioral observation, typically by remote video monitoring (High-speedDigital Video Recording System EVS-DSX-16000DVD-H with CCD cameras, EpicSystems Inc., St. Louis, Mo., USA) followed by blinded off-line scoringof video recordings. Rats were observed for one hour, and the number ofyawns, grooming bouts and PEs were recorded in 10-minute bins.

PE results from studies with 1.0-3.0 mg/kg subcutaneously of Examples43, 46 and 67, along with Ref Ex 2, are shown in Table 3 below,expressed as total number of PE/rat, mean±SEM (n=7-11) and number ofrats/group showing at least one erection over the study period (%responders). Corresponding vehicle controls (3.2% mannitol/50 mM Trisbuffer) were run in parallel.

TABLE 3 Dose Total number of Treatment (mg/kg, s.c.) n PE/rat 0-1 h %Responders Vehicle 11 1.0 ± 0.27 64 Ref Ex 2 3.0 7 6.0 ± 0.44 100 Ex 431.0 7 1.7 ± 0.36 29 3.0 7 6.0 ± 0.76 100 Ex 46 1.0 7 4.9 ± 1.1  100 3.07 4.4 ± 1.1  86 Ex 67 1.0 7 2.3 ± 0.42 86 3.0 7 3.3 ± 1.0  100

8.0 High and Low Density hMC4-R Functional Assay Results

Exemplified peptides were tested using the high and low density hMC4-Rfunctional assay as described in Section 7.3 above, with the results asshown in Table 4 below. Table 4 includes both single point values andmean values.

TABLE 4 0.1 ng/mL Doxycycline 10 ng/mL Doxycycline Intrinsic IntrinsicCompound EC₅₀ (nM) Activity (%) EC₅₀ (nM) Activity (%) Example 1 0.34 910.045 96 Example 2 0.56 36 0.095 99 Example 3 0.07 28 0.16 100 Example 4— 11 0.245 100 Example 5 — 5 0.38 92 Example 6 37 29 2 100 Example 7 0.348 0.135 100 Example 8 48 71 0.55 87 Example 9 0.09 91 0.035 89 Example10 0.45 77 0.17 94 Example 11 0.065 83 0.06 90 Example 12 34 64 0.5 97Example 13 0.125 85 0.06 96 Example 14 8 16 0.4 93 Example 15 0.165 160.2 90 Example 16 0.15 28 0.16 97 Example 17 0.95 64 0.12 95 Example 180.14 83 0.085 97 Example 19 0.42 35 0.22 97 Example 20 — 0.5 0.75 53Example 21 — 8 0.5 97 Example 22 3 78 0.2 93 Example 23 2 17 0.55 95Example 24 0.7 15 0.45 99 Example 25 75 23 2 94 Example 26 0.125 74 0.1290 Example 27 0.7 37 0.33 97 Example 28 29 35 0.6 94 Example 29 3 14 0.8100 Example 30 — 2 25 9 Example 31 7 33 0.313 98 Example 32 18 34 0.54396 Example 33 1 41 0.153 98 Example 34 1 79 0.137 97 Example 35 — 6 1 84Example 36 — 8 1 89 Example 37 — 6 0.55 90 Example 38 2 31 0.258 97Example 39 — 4 1 79 Example 40 — 8 0.5 93 Example 41 — 5 0.8 84 Example42 — 9 0.5 84 Example 43 1 43 0.177 100 Example 44 11 22 0.617 99Example 45 — 12 0.45 87 Example 46 0.666 40 0.173 97 Example 47 0.31 360.373 100 Example 48 — 6 4 93 Example 49 2 74 0.235 93 Example 50 0.2194 0.085 94 Example 51 0.64 22 0.4 95 Example 52 0.325 31 0.2 99 Example53 0.13 86 0.13 90 Example 54 0.075 56 0.075 92 Example 55 10 28 0.8 98Example 56 5 41 0.3 98 Example 57 0.25 89 0.095 100 Example 58 5 67 0.25100 Example 59 0.6 90 0.1 98 Example 60 0.45 62 0.14 95 Example 61 — 60.95 89 Example 62 16 23 0.47 84 Example 63 0.37 30 0.157 93 Example 641 57 0.08 90 Example 65 1 58 0.085 93 Example 66 5 32 0.265 88 Example67 1 38 0.14 99 Example 68 0.85 55 0.075 91 Example 69 0.25 76 0.095 92Example 70 0.4 64 0.1 91 Example 71 1 86 0.085 91 Example 72 3 11 0.3584 Example 73 0.587 86 0.068 102 Example 74 0.943 68 0.11 100 Example 750.65 43 0.125 87 Example 76 3 53 0.135 85 Example 77 3 34 0.155 80Example 78 2 48 0.2 89 Example 79 4 48 0.2 85 Example 80 6 13 0.65 86Example 81 4 23 0.45 85 Example 82 2 79 0.095 95 Example 83 0.75 780.075 94 Example 84 0.633 78 0.09 97 Example 85 15 36 0.707 94 Example86 9 36 0.483 96 Example 87 1 64 0.065 100 Example 88 3 15 0.35 89Example 89 6 17 87 Example 90 4 31 0.283 96 Example 91 1 18 0.265 96Example 92 0.1 81 0.04 89 Example 93 0.3 44 0.17 97 Example 94 0.24 320.09 92 Example 95 1 42 0.125 100 Example 96 2 24 0.235 100 Example 970.8 49 0.1 100 Example 98 0.45 47 0.14 100 Example 99 0.7 32 0.14 94Example 100 6 27 0.27 93 Example 101 0.95 36 0.14 93 Example 102 18 300.725 97 Example 103 1 32 0.13 100 Example 104 191 16 10 100 Example 1053 35 0.15 94 Example 106 3 113 77 Example 107 4 64 Example 108 3 9 59Example 109 2 269 69 Example 110 0.6 84 0.06 105 Example 111 0.575 810.053 104 Example 112 0.39 83 0.07 101 Example 113 0.72 80 0.065 100Example 114 1 73 0.185 117 Example 115 0.8 54 0.17 108 Example 116 0.6877 0.095 104 Example 117 1 50 0.18 112

It is believed that peptides of the invention exhibiting an intrinsicactivity of equal to or greater than about 0.1 (10%), such as greaterthan 0.2 (20%) or greater than about 0.3 (30%) or greater than about 0.4(40%) or greater than about 0.5 (50%) or greater than about 0.6 (60%) %)or greater than about 0.7 (70%), or greater than about 0.8 (80%), orgreater than about 0.9 (90%), or equal to or greater than about 1.0(100%), in the above described high density hMC4-R system (i.e.HEK293TRexMC4R cells treated with 10 ng/ml doxycycline), based onmaximal stimulation of adenylyl cyclase achievable by the compound inthe same high density hMC4-R system where the maximal stimulationachieved by α-MSH or NDP-α-MSH is designated as an intrinsic activity of1.0 (100%), provide agonist (full or partial) activity on the MC4receptor.

A majority of the peptides of the Examples which were tested in theabove described high density hMC4-R system (i.e. HEK293TRexMC4R cellstreated with 10 ng/ml doxycycline) gave in said assay EC₅₀ values forcAMP production of less than 0.05 μM, such as less than 0.01 μM, and inparticular less than 0.001 μM.

As seen from Table 4, peptides of the invention may lack intrinsicactivity in the low density hMC4-R system and be full agonists in thehigh density hMC4-R system.

As further seen from Table 4, peptides of the invention may be partialagonists in the above described low density hMC4-R system and fullagonists in the above described high density hMC4-R system.

As further seen from Table 4, peptides of the invention may be fullagonists in both the low density hMC4-R system and in the high densityhMC4-R system.

Without being bound by any theory, it is believed that peptides thateither lack intrinsic activity or are partial agonists in the abovedescribed low density hMC4-R system (i.e. peptides having intrinsicactivity of from 0 to 70% in the low density hMC4-R system) and arepartial or full agonists in the above described high density hMC4-Rsystem (i.e. peptides having intrinsic activity of equal to or above10%, or above 70%, in the high density hMC4-R system) will provide no ora reduced level of sexual effects resulting from MC4 receptoractivation, e.g. penile erection, compared to peptides being fullagonists in both the low and the high density hMC4-R systems. Asexplained above, these sexual effects are considered unwantedside-effects upon treatment of energy homeostasis and metabolismrelated, food intake related and/or energy balance and body weightrelated diseases, disorders and/or conditions.

It is thus believed that peptides according to the invention possesspromising MC4 receptor affinity, efficacy and potency to be useful fortreatment of diseases, disorders and/or conditions responsive toactivation of the MC4 receptor, in particular energy homeostasis andmetabolism related (e.g. diabetes), food intake related and/or energybalance and body weight related diseases, disorders and/or conditions,including obesity, overweight and diseases, disorders and/or conditionsassociated with obesity and/or overweight, such as type 2 diabetes andmetabolic syndrome.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverall such modifications and equivalents.

1. A cyclic peptide of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R₁ is —NH—C(═O)—or —C(═O)—NH—; R₂ is —H or —CH₂—, and if R₂ is —CH₂— forms with R₃ apyrrolidine ring, the pyrrolidine ring optionally substituted with —OH;R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwise R₃ is selected from

R_(4a), R_(4b) and R_(4c) are each independently selected from hydrogen,halo, (C₁-C₁₀)alkyl-halo, (C₁-C₁₀)alkyl-dihalo, (C₁-C₁₀)alkyl-trihalo,(C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy, (C₁-C₁₀)alkylthio, aryl, aryloxy, nitro,nitrile, sulfonamide, amino, monosubstituted amino, disubstituted amino,hydroxy, carboxy, and alkoxy-carbonyl, on the proviso that at least oneof R_(4a), R_(4b) and R_(4c) is not hydrogen; R₅ is —OH or—N(R_(6a))(R_(6b)); R_(6a) and R_(6b) are each independently H or a C₁to C₄ linear, branched or cyclic alkyl chain; R₇ is —H or —C(═O)—NH₂; wis in each instance independently 0 to 5; x is 1 to 5; y is 1 to 5; andz is in each instance independently 1 to
 5. 2. The cyclic peptide ofclaim 1 which is of Formula (II):

or a pharmaceutically acceptable salt thereof.
 3. The cyclic peptide ofclaim 1, wherein R₁ is —C(═O)—NH—, x is 2 and y is
 3. 4. The cyclicpeptide of claim 1, wherein R₁ is —NH—C(═O)—, x is 3 and y is
 2. 5. Thecyclic peptide of claim 1, wherein R₂ is H or —CH₂—, and if R₂ is —CH₂—forms with R₃ a pyrrolidine ring, the pyrrolidine ring optionallysubstituted with —OH; and R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwiseR₃ is selected from


6. The cyclic peptide of claim 1, wherein R₂ is H or —CH₂—, and if R₂ is—CH₂— forms with R₃ a pyrrolidine ring, the pyrrolidine ring optionallysubstituted with —OH; and R₃ is —(CH₂)₂— if R₂ is —CH₂—, and otherwiseR₃ is selected from


7. The cyclic peptide of claim 1, wherein R₂ is H, R₃ is selected from


8. The cyclic peptide of claim 1, wherein R₂ is —CH₂— and R₃ is—(CH₂)₂—, R₂ and R₃ together forming an unsubstituted pyrrolidine ring.9. The cyclic peptide of claim 1, wherein at least one of R_(4a), R_(4b)and R_(4c) is independently selected from


10. The cyclic peptide of claim 1, wherein at least one of R_(4a),R_(4b) and R_(4c) is independently selected from hydrogen, halo,(C₁-C₄)alkyl-halo, (C₁-C₄)alkyl-dihalo, (C₁-C₄)alkyl-trihalo,(C₁-C₄)alkyl, (C₁-C₄)alkoxy, nitro, nitrile, amino and hydroxy, on theproviso that at least one of R_(4a), R_(4b) and R_(4c) is not hydrogen.11. The cyclic peptide of claim 1 wherein R_(4a) is in the 4 positionand is —C≡N and R_(4b) and R_(4c) are each H.
 12. The cyclic peptide ofclaim 1 wherein R_(4a) is in the 4 position and is —F and R_(4b) andR_(4c) are each H.
 13. A cyclic peptide selected from: (SEQ ID NO: 48)Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 51)Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 74)Ac-Arg-cyclo(Glu-Asn-D-Phe(4-F)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 90)Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)- OH; (SEQ ID NO: 91)Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)- OH; (SEQ ID NO: 97)Ac-Arg-cyclo(Glu-Gln-D-Phe(4-F)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 44)Ac-Arg-cyclo(Glu-Pro-D-Phe(4-CN)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 72)Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)- NH₂; (SEQ ID NO: 107)Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-OH;

or a pharmaceutically acceptable salt of any of the foregoing.
 14. Apharmaceutical composition comprising a cyclic peptide or apharmaceutically acceptable salt thereof of claim 1 and apharmaceutically acceptable carrier. 15-17. (canceled)
 18. A method oftreating a disease, disorder and/or condition responsive to activationof the MC4 receptor comprising administering a therapeutically effectiveamount of a peptide according to claim 1 to a patient in need thereof.19. A method of treating diabetes, obesity, overweight and/or diseases,disorders and/or conditions associated with obesity and/or overweight,including insulin resistance; impaired glucose tolerance; type 2diabetes; metabolic syndrome; dyslipidemia; hyperlipidemia;hypertension; heart disorders; cardiovascular disorders; non-alcoholicfatty liver disease; joint disorders; secondary osteoarthritis;gastroesophageal reflux; sleep apnea; atherosclerosis; stroke; macro andmicro vascular diseases; steatosis; gall stones; and gallbladderdisorders comprising administering a therapeutically effective amount ofa peptide according to claim 1 to a patient in need thereof.
 20. Amethod of reducing food intake, body weight and/or body weight gaincomprising administering a pharmacologically effective amount of apeptide according to claim 1 to an individual in need thereof.